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T-regulatory cells (Tregs) are essential for immune tolerance, and animal studies implicate their dysfunction in type 1 diabetes (T1D) pathogenesis. Tregs require interleukin-2 (IL-2) for their suppressive function, and variants in IL-2/IL-2R pathway genes have been associated with T1D. We previously reported that recent-onset T1D subjects have an increased population of FOXP3lo Tregs that secrete the pro-inflammatory cytokine, interleukin-17 (IL-17). We hypothesize that IL-2 signaling defects may drive T1D development by skewing protective Tregs towards an inflammatory Th17 phenotype. Overall, we found that the proportion of FOXP3+IL-17+ cells in T1D subjects pre-diagnosis was unchanged compared with healthy controls. However, stratification by IL2RA single-nucleotide polymorphisms revealed that T1D subjects with the rs3118470 CC risk variant have Tregs with IL-2 signaling defects and an increased proportion of FOXP3+IL-17+ cells before diagnosis. These data suggest a potential mechanism for genetically controlled loss of Treg function via dysfunctional IL-2 signaling in T1D.

Highlights • Respanel is a laboratory-developed PCR test panel for common respiratory pathogens. • Strip-tube array-based assay, designed for convenience and minimal hands-on time. • Specificity and accuracy of Respanel is ≥95% against two commercial assays. • Sensitivity of Respanel is 94% and 88% against two commercial assays., Commercial multiplex assays, built on different chemistries and platforms are widely available for simultaneous detection of pathogens that cause respiratory infections. However, these tests are often difficult to implement in a resource limited setting because of high cost. In this study, we developed and validated a method for simultaneous testing of common respiratory pathogens (Respanel) by real-time PCR in a convenient, strip-tube array format. Primers and probes for sixteen PCR assays were selected from the literature or newly designed. Following optimization of individual PCR assays, strip-tube arrays were prepared by dispensing primer-probe mixes (PPM) into two sets of 8-tube strips. Nucleic acid extracts from specimens were mixed with PCR master mix, and dispensed column-wise into 2 × 8-wells of a 96-well plate. PPMs from strip-tubes were then added to the wells using a multichannel pipette for real-time PCR. Individual PCR assays were optimized using previously known specimens (n = 394) with 91%–100% concordance with culture, DFA or PCR results. Respanel was then tested in a routine manner at two different sites using specimens (n = 147) previously tested by Qiagen Resplex I&II or Fast-Track Diagnostics Respiratory Pathogens 21 assays. The sensitivity, specificity and accuracy of Respanel were 94%, 95% and 95%, respectively, against Resplex and 88%, 100% and 99%, respectively, against FTDRP21. Respanel detected more pathogens (p

T regulatory cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and nonharmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require these cells to be near their targets of suppression; however, it is not known how Treg-to-target proximity is controlled. Here, we found that Tregs attract CD4+ and CD8+ T cells by producing chemokines. Specifically, Tregs produced both CCL3 and CCL4 in response to stimulation, and production of these chemokines was critical for migration of target T cells, as Tregs from Ccl3–/– mice, which are also deficient for CCL4 production, did not promote migration. Moreover, CCR5 expression by target T cells was required for migration of these cells to supernatants conditioned by Tregs. Tregs deficient for expression of CCL3 and CCL4 were impaired in their ability to suppress experimental autoimmune encephalomyelitis or islet allograft rejection in murine models. Moreover, Tregs from subjects with established type 1 diabetes were impaired in their ability to produce CCL3 and CCL4. Together, these results demonstrate a previously unappreciated facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their therapeutic effect in autoimmunity and transplantation.

C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in monocyte-derived dendritic cells, in which it strongly co-localized with human leukocyte antigen class II. Treatment of these professional antigen-presenting cells with vitamin D, a key protective environmental factor in multiple sclerosis, downmodulated CLEC16A in parallel with human leukocyte antigen class II. Knockdown of CLEC16A in distinct types of model and primary antigen-presenting cells resulted in severely impaired cytoplasmic distribution and formation of human leucocyte antigen class II-positive late endosomes, as determined by immunofluorescence and electron microscopy. Mechanistically, CLEC16A participated in the molecular machinery of human leukocyte antigen class II-positive late endosome formation and trafficking to perinuclear regions, involving the dynein motor complex. By performing co-immunoprecipitations, we found that CLEC16A directly binds to two critical members of this complex, RILP and the HOPS complex. CLEC16A silencing in antigen-presenting cells disturbed RILP-mediated recruitment of human leukocyte antigen class II-positive late endosomes to perinuclear regions. Together, we identify CLEC16A as a pivotal gene in multiple sclerosis that serves as a direct regulator of the human leukocyte antigen class II pathway in antigen-presenting cells. These findings are a first step in coupling multiple sclerosis-associated genes to the regulation of the strongest genetic factor in multiple sclerosis, human leukocyte antigen class II.

IL-17 plays critical roles in host defenses, combating bacterial and fungal infections, as well as the pathogenesis of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). The signaling adaptor SAP is essential for normal immune homeostasis and mutations within SH2D1A, the locus encoding this protein, result in serious and sometimes fatal syndromes, including X-linked lymphoproliferative disease and severe cases of common variable immunodeficiency. However, the precise cellular basis of how SAP deficiency contributes to immune dysfunction remains incompletely understood. In this study, we found that CD4 and CD8 T cells lacking SAP had a diminished capacity to differentiate into IL-17–producing Th17 and T cytotoxic (Tc17) cells relative to wild-type lymphocytes. The use of costimulating SLAM Abs was found to augment the differentiation of IL-17–secreting effectors in wild-type but not Sh2d1a−/− splenic T cells under IL-17–polarizing conditions. In addition, SAP’s regulation of IL-17–secreting T cells was shown to be a T cell–intrinsic role, as purified naive Sh2d1a−/− CD4 and CD8 T cells were inherently defective at converting into Th17 and Tc17 cells in vitro and in vivo. Furthermore, Sh2d1a−/− mice were protected from EAE and exhibited greatly decreased numbers of CNS-infiltrating Th17 and Tc17 effector T cells and reduced disease severity. Collectively, these results suggest that SLAM–SAP signaling drives the differentiation and function of Th17 and Tc17 cells in vitro and in vivo and contributes to the pathogenesis of autoimmunity in EAE.

Individuals with X-linked lymphoproliferative disease lack invariant natural killer T (iNKT) cells and are exquisitely susceptible to Epstein-Barr virus (EBV) infection. To determine whether iNKT cells recognize or regulate EBV, resting B cells were infected with EBV in the presence or absence of iNKT cells. The depletion of iNKT cells increased both viral titers and the frequency of EBV-infected B cells. However, EBV-infected B cells rapidly lost expression of the iNKT cell receptor ligand CD1d, abrogating iNKT cell recognition. To determine whether induced CD1d expression could restore iNKT recognition in EBV-infected cells, lymphoblastoid cell lines (LCL) were treated with AM580, a synthetic retinoic acid receptor-α agonist that upregulates CD1d expression via the nuclear protein, lymphoid enhancer-binding factor 1 (LEF-1). AM580 significantly reduced LEF-1 association at the CD1d promoter region, induced CD1d expression on LCL, and restored iNKT recognition of LCL. CD1d-expressing LCL elicited interferon γ secretion and cytotoxicity by iNKT cells even in the absence of exogenous antigen, suggesting an endogenous iNKT antigen is expressed during EBV infection. These data indicate that iNKT cells may be important for early, innate control of B cell infection by EBV and that downregulation of CD1d may allow EBV to circumvent iNKT cell-mediated immune recognition.

The stability of pathogen-specific DNA or RNA amplification targets in clinical samples following short-term storage at room temperature, 4°C, and −80°C was assessed by real-time PCR. In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, irrespective of storage temperature. In unextracted samples, temperature-dependent loss of targets ( P < 0.05) was observed in serum and cerebrospinal fluid specimens, while no changes were observed for EDTA blood samples.

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) probably play the major role in controlling HIV replication. However, the value of adoptive transfer of HIV-specific CTL expanded in vitro to HIV+ patients has been limited: this contrasts with the success of CTL therapy in treating or preventing Epstein-Barr virus and cytomegalovirus disease after bone marrow transplantation (BMT). We investigated the fate of expanded HIV-specific CTL clones in vivo following adoptive transfer to a patient with acquired immunodeficiency syndrome (AIDS). Two autologous CTL clones specific for HIV Gag and Pol were expanded to large numbers (>109) in vitro and infused into an HIV-infected patient whose viral load was rising despite antiretroviral therapy. The fate of one clone was monitored by staining peripheral blood mononuclear cells (PBMCs) with T-cell receptor–specific tetrameric major histocompatibility complex (MHC)-peptide complexes. Although the CTL transfer was well tolerated, there were no significant changes in CD4 and CD8 lymphocyte counts and virus load. By tracking an infused clone using soluble MHC-peptide complexes, we show that cells bearing the Gag-specific T-cell receptors were rapidly eliminated within hours of infusion through apoptosis. Thus, the failure of adoptively transferred HIV-specific CTL to reduce virus load in AIDS may be due to rapid apoptosis of the infused cells, triggered by a number of potential mechanisms. Further trials of adoptive transfer of CTL should take into account the susceptibility of infused cells to in vivo apoptosis.

Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%; P < 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome ( P < 0.05) and improved the sensitivity of pathogen detection ( P < 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.

Manipulation of regulatory T cell (Treg) migration by islet expression of the chemokine CCL22 prevents diabetes in NOD mice and delays recurrent autoimmunity in syngeneic islet transplants. We sought to determine whether attracting Tregs with CCL22 also prevents islet allograft rejection. Isolated Bl/6 mouse islets were transduced overnight with adenovirus expressing CCL22 (Ad-CCL22) downstream of the CMV promoter. Islets were transplanted under the renal capsule of Balb/c recipients made diabetic by streptozotocin. To assess immunologic tolerance, graft-bearing kidneys from recipients of CCL22-expressing islet grafts were removed, and mice received a second transplant of naive islets from the same donor strain or third-party islets into the contralateral kidney. Adenoviral expression of CCL22 conferred prolonged protection of islet allografts in MHC-mismatched, diabetic recipients, maintaining normoglycemia in 75% of recipients for at least 80 days. Increased frequency of Treg cells was observed in islet grafts transduced with Ad-CCL22 compared with untreated grafts. Normoglycemic recipients of CCL22-expressing islet grafts showed complete absence of antidonor antibodies and no lymphocyte proliferation after exposure to donor splenocytes. After removal of the primary graft at day 80, mice that received a second transplant with untreated islets from the same donor strain did not reject the grafts, suggesting the development of tolerance. Expression of CCL22 recruits Treg cells to transplanted islets, prevents activation of alloreactive T-cells and islet allograft failure and induces alloantigen-specific tolerance. Manipulation of Treg cells by CCL22 in transplanted islets may be a novel therapeutic strategy for diabetes.

Citation Eastabrook GDM, Hu Y, Tan R, Dutz JP, MacCalman CD, von Dadelszen P. Decidual NK cell-derived conditioned medium (dNK-CM) mediates VEGF-C secretion in extravillous cytotrophoblasts. Am J Reprod Immunol 2012; 67: 101–111 Problem The regulatory mechanisms involved in VEGF-C secretion by trophoblasts during placentation are poorly understood. We investigated whether or not decidual natural killer cell conditioned medium (dNK-CM) stimulated VEGF-C secretion in the extravillous cytotrophoblast (EVT) cell line HTR8/SVneo. Method of Study The effects of dNK-CM and recombinant IFN-γ on VEGF-C induction by HTR8/SVneo were studied in the absence or presence of IFN-γ or its receptor blocking antibodies, p38 inhibitor (SB202190), JAK inhibitor (JAK inhibitor-1, JI-1), and on STAT1 knockdown HTR8/SVneo. VEGF-C was quantified by ELISA. FACS was used to investigate the phosphorylations of Tyr701 or Ser727 of STAT1 on stimulated HTR8/SVneo. Results dNK-CM facilitated VEGF-C secretion by HTR8/SVneo. IFN-γ and IFN-γR1 or IFN-γR2 blocking antibodies reduced both dNK-CM- and IFN-γ-induced VEGF-C secretion. Phosphorylations on Tyr701 or Ser727 of STAT1 were elevated upon stimulation. Secretion of VEGF-C was reduced by treatment with SB202190, JI-1, or STAT1 knockdown by siRNA. Conclusion VEGF-C production by trophoblasts is regulated by soluble factors secreted by dNK through p38 and JAK-STAT1 pathways.

Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells. Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22. Here, we demonstrate that production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice. In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes. CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the CD4+ T cell population near islets, and decreased the frequency of circulating autoreactive CD8+ T cells and CD8+ IFN-γ–producing T cells. The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody. Our results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells. CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes.

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downreglation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.

Made available in DSpace on 2013-09-27T14:54:12Z (GMT). No. of bitstreams: 1 WOS000288773100013.pdf: 1615338 bytes, checksum: 809906f90b3beafd045fea12e494091f (MD5) Previous issue date: 2011-04-10 Made available in DSpace on 2013-09-30T18:23:19Z (GMT). No. of bitstreams: 1 WOS000288773100013.pdf: 1615338 bytes, checksum: 809906f90b3beafd045fea12e494091f (MD5) Previous issue date: 2011-04-10 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:37:48Z No. of bitstreams: 1 WOS000288773100013.pdf: 1615338 bytes, checksum: 809906f90b3beafd045fea12e494091f (MD5) Made available in DSpace on 2014-05-20T13:37:48Z (GMT). No. of bitstreams: 1 WOS000288773100013.pdf: 1615338 bytes, checksum: 809906f90b3beafd045fea12e494091f (MD5) Previous issue date: 2011-04-10 NIH Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) American Cancer Society UPCI Core Facility Cancer Center KSHV LANA1, a latent protein expressed during chronic infection to maintain a viral genome, inhibits major histocompatibility complex class I (MHCI) peptide presentation in cis as a means of immune evasion. Through deletional cloning, we localized this function to the LANA1 central repeat 1 (CR1) subregion. Other CR subregions retard LANA1 translation and proteasomal processing but do not markedly inhibit LANA1 peptide processing by MHC I. Inhibition of proteasomal processing ablates LANA1 peptide presentation. Direct expression of LANA1 within the endoplasmic reticulum (ER) overcomes CR1 inhibition suggesting that CR1 acts prior to translocation of cytoplasmic peptides into the ER. By physically separating CR1 from other subdomains, we show that LANA1 evades MHC I peptide processing by a mechanism distinct from other herpesviruses including Epstein-Barr virus (EBV). Although LANA1 and EBV EBNA1 are functionally similar, they appear to use different mechanisms to evade host cytotoxic T lymphocyte surveillance. (C) 2011 Elsevier B.V. All rights reserved. Univ Pittsburgh, Inst Canc, Canc Virol Program, Pittsburgh, PA 15213 USA São Paulo State Univ, Botucatu Sch Med, Dept Pathol, São Paulo, Brazil Univ British Columbia, British Columbia Childrens Hosp, Dept Pathol & Lab Med, Vancouver, BC V5Z 1M9, Canada Univ Pittsburgh, Med Ctr, Dept Otolaryngol, Pittsburgh, PA 15213 USA Univ Pittsburgh, Med Ctr, Dept Immunol, Pittsburgh, PA 15213 USA Univ Pittsburgh, Inst Canc, Canc Immunol Program, Pittsburgh, PA 15213 USA São Paulo State Univ, Botucatu Sch Med, Dept Pathol, São Paulo, Brazil NIH: NCI121930 CAPES: BEX1049/05-4 UPCI: P30 CA047904

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1−/− 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.

Natural killer T (NKT) cells are innate-like lymphocytes that recognize lipid antigens and have been shown to enhance B-cell activation and antibody production. B cells typically recruit T-cell help by presenting internalized antigens recognized by their surface antigen receptor. Here, we demonstrate a highly efficient means whereby human B cells present lipid antigens to NKT cells, capturing the antigen using apolipoprotein E (apoE) and the low-density lipoprotein receptor (LDL-R). ApoE dramatically enhances B-cell presentation of alpha-galactosylceramide (αGalCer), an exogenous CD1d presented antigen, inducing activation of NKT cells and the subsequent activation of B cells. B cells express the LDL-R on activation, and the activation of NKT cells by B cells is completely LDL-R dependent, as shown by blocking experiments and the complete lack of presentation when using apoE2, an isoform of apoE incapable of LDL-R binding. The dependence on apoE and the LDL-R is much more pronounced in B cells than we had previously seen in dendritic cells, which can apparently use alternate pathways of lipid antigen uptake. Thus, B cells use an apolipoprotein-mediated pathway of lipid antigen presentation, which constitutes a form of innate help for B cells by NKT cells.

X-linked lympho-proliferative (XLP) is an immunodeficiency condition caused by mutation or deletion of the gene encoding the adaptor protein SAP/SH2D1A. Besides defects in T cell and NK cell function and an absence of NKT cells, XLP can also manifest as lymphomas resulting primarily from uncontrolled B cell proliferation upon acute infection by Epstein-Barr virus. While it has been demonstrated that SAP regulates the functions of T cells and NK cells through the SLAM family of immunoreceptors, its role in B cells has not been defined. Here we show that SAP forms a ternary complex with the kinase Lyn and the inhibitory IgG Fc receptor FcgammaRIIB to regulate B cell proliferation and survival. SAP binds directly and simultaneously to the Lyn SH3 domain and an Immuno-receptor Tyrosine-based Inhibitory Motif (ITIM) in FcgammaRIIB, resulting in the activation of the latter. Moreover, SAP associates with FcgammaRIIB in mouse splenic B cells and promotes its tyrosine phosphorylation. Expression of SAP in the A20 B cell line led to a marked reduction in Blnk phosphorylation, a decrease in Akt activation, and a near-complete ablation of phosphorylation of the MAP kinases Erk1/2, p38 and JNK upon colligation of FcgammaRIIB with the B cell receptor (BCR). In contrast, an XLP-causing SAP mutant was much less efficient in eliciting these effects in B cells. Furthermore, compared to A20 cells, SAP transfectants displayed a significantly reduced rate of proliferation and an increased sensitivity to activation-induced cell death. Collectively these data identify an intrinsic function for SAP in inhibitory signaling in B cells and suggests that SAP may play an important role in balancing positive versus negative immune responses.

Pre-eclampsia is often associated with inadequate cytotrophoblast invasion and remodelling of the uterine spiral arteries. Examining a first trimester, 2D in vitro explant culture model which mimics in vivo placentation, including trophoblast column formation and extravillous cytotrophoblast (EVT) migration, we previously suggested that excessive maternal decidual natural killer cell interferon (IFN-gamma) limits EVT migration. Types-1 and -2 insulin-like growth factor (IGF-1, IGF-2) are trophic for EVT, act through their surface receptors, IGFR-1 and IGFR-2, and are regulated by the IGF-binding proteins (IGFBPs). Could the observed IFN-gamma-mediated inhibition of EVT outgrowth and migration be related to either expression changes of IGF-1 or IGF-2, their receptors, their binding proteins, or apoptosis? Using the 2D explant culture model, we examined the effect of IFN-gamma exposure on IGF-1 and -2, IGFR-1 and -2, IGFBPs and apoptosis. IFN-gamma relatively increased IGF-1 and -2 secretion. In EVT, IFN-gamma decreased IGFR-2, but not IGFR-1 expression. IGBP-2, -3 and -4 production were not influenced by IFN-gamma. IFN-gamma induced trophoblast apoptosis measured by the highly sensitive M30 neo-epitope, but not caspase 3 activity, in conditioned medium and EVT cell lysates. The observed IFN-gamma-mediated EVT migration inhibition may occur through the down-regulation of IGFR-2 and subtle induction of EVT apoptosis.

NK cells and CD8+ T cells bind MHC-I molecules using distinct topological interactions. Specifically, murine NK inhibitory receptors bind MHC-I molecules at both the MHC-I H chain regions and β2-microglobulin (β2m) while TCR engages MHC-I molecules at a region defined solely by the class I H chain and bound peptide. As such, alterations in β2m are not predicted to influence functional recognition of MHC-I by TCR. We have tested this hypothesis by assessing the capability of xenogeneic β2m to modify the interaction between TCR and MHC-I. Using a human β2m-transgenic C57BL/6 mouse model, we show that human β2m supports formation and expression of H-2Kb and peptide:H-2Kb complexes at levels nearly equivalent to those in wild-type mice. Despite this finding, the frequencies of CD8+ single-positive thymocytes in the thymus and mature CD8+ T cells in the periphery were significantly reduced and the TCR Vβ repertoire of peripheral CD8+ T cells was skewed in the human β2m-transgenic mice. Furthermore, the ability of mouse β2m-restricted CTL to functionally recognize human β2m+ target cells was diminished compared with their ability to recognize mouse β2m+ target cells. Finally, we provide evidence that this effect is achieved through subtle conformational changes occurring in the distal, peptide-binding region of the MHC-I molecule. Our results indicate that alterations in β2m influence the ability of TCR to engage MHC-I during normal T cell physiology.

Recently, it has been shown that human beta(2)-microglobulin (h-beta(2)m) blocks the association between the NK cell inhibitory receptor Ly49C and H-2K(b). Given this finding, we therefore sought to assess the immunobiology of NK cells derived from C57BL/6 (H-2(b)) mice expressing exclusively h-beta(2)m. Initial analysis revealed that the Ly49C expression profile of NK cells from h-beta(2)m(+) mice was modified, despite the fact that H-2K(b) expression was normal in these mice. Moreover, the NK cells were not anergic in that IL-2 treatment of h-beta(2)m(+) NK cells in vitro enabled efficient lysis of prototypic tumor cell lines as well as of syngeneic h-beta(2)m(+) lymphoblasts. This loss of self-tolerance appeared to correlate with the activation status of h-beta(2)m(+) NK cells because quiescent h-beta(2)m(+) transplant recipients maintained h-beta(2)m(+) grafts but polyinosine:polycytidylic acid-treated recipients acutely rejected h-beta(2)m(+) grafts. NK cell reactivity toward h-beta(2)m(+) targets was attributed to defective Ly49C interactions with h-beta(2)m:H-2K(b) molecules. With regard to NK cell regulatory mechanisms, we observed that h-beta(2)m:H-2K(b) complexes in the cis-configuration were inefficient at regulating Ly49C and, furthermore, that receptor-mediated uptake of h-beta(2)m:H-2K(b) by Ly49C was impaired compared with uptake of mouse beta(2)m:H-2K(b). Thus, we conclude that transgenic expression of h-beta(2)m alters self-MHC class I in such a way that it modulates the NK cell phenotype and interferes with regulatory mechanisms, which in turn causes in vitro-expanded and polyinosine:polycytidylic acid-activated NK cells to be partially self-reactive similar to what is seen with NK cells derived from MHC class I-deficient mice.

Background Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar ® BKV PCR Kit. Method Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar ® BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. Results The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8 × 10 2 , 3 × 10 3 and 3.5 × 10 2 genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar ® BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were ® BKV PCR assay, were highly specific when tested against a panel of external proficiency specimens containing both BK and JC viruses. All assays, except the VP1MOD assay determined BK viral load in proficiency specimens within the same log values. With reference to results obtained by RealStar ® BKV PCR assay, the sensitivity and specificity of different assays tested in 116 serum specimens submitted for BK viral load assay were 91% and 97% for VP1 assay, 88% and 97% for VP1MOD assay, and 97% and 98% for BKLTA assay, respectively. BK Viral load in positive specimens determined by various assays was highly correlated (R 2 > 0.97), based on linear regression analysis. Conclusions The performance characteristics of the newly designed, BKLTA assay were highly comparable to RealStar ® BKV PCR assay, and can be used for routine detection and viral load monitoring of BKV in a cost-effective manner.

Invariant natural killer T (iNKT) cells are a highly conserved subset of unconventional T lymphocytes that express a canonical, semi-invariant T cell receptor (TCR) and surface markers shared with the natural killer cell lineage. iNKT cells recognize exogenous and endogenous glycolipid antigens restricted by non-polymorphic CD1d molecules, and are highly responsive to the prototypical agonist, α-galactosylceramide. Upon activation, iNKT cells rapidly coordinate signaling between innate and adaptive immune cells through the secretion of proinflammatory cytokines, leading to the maturation of antigen-presenting cells and expansion of antigen-specific CD4+ and CD8+ T cells. Because of their potent immunoregulatory properties, iNKT cells have been extensively studied and are known to play a pivotal role in mediating immune responses against microbial pathogens including viruses. Here, we review evidence that herpesviruses manipulate CD1d expression to escape iNKT cell surveillance and establish lifelong latency in humans. Collectively, published findings suggest that iNKT cells play critical roles in anti-herpesvirus immune responses and could be harnessed therapeutically to limit viral infection and viral-associated disease.

Abnormal placentation results in either inadequate (consequences: recurrent miscarriage, intrauterine growth restriction, and preeclampsia) or overzealous (consequences: placenta accreta, increta, and percreta) placentation. NK cells dominate in first trimester decidua and probably control extravillous cytotrophoblast (EVT) invasion. We examined this interaction in a novel way, using NK cells and villous explants from concordant first trimester pregnancies cocultured using a new collagen (two-dimensional) model of placentation. Decidual NK (dNK) cells exerted contact-independent inhibition of normal cytotrophoblast migration, associated with changes in the cytotrophoblast expression of metalloproteases-2 and -9, and plasminogen activator inhibitor-1. dNK cells did not affect EVT proliferation and apoptosis, and cell column formation. dNK cell effects were partially reversed by neutralizing Abs against IFN-γ. We provide ex vivo human evidence of a direct role for dNK in modulating EVT differentiation as they form columns and then migrate from anchoring villi.

Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic beta-cells by cytotoxic T-lymphocytes (CTLs). In humans, few beta-cell epitopes have been reported, thereby limiting the study of beta-cell-specific CTLs in type 1 diabetes. To identify additional epitopes, HLA class I peptide affinity algorithms were used to identify a panel of peptides derived from the beta-cell proteins islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, insulinoma-associated antigen 2 (IA-2), and phogrin that were predicted to bind HLA-A*0201. Peripheral blood mononuclear cells from 24 HLA-A*0201 recent-onset type 1 diabetic patients and 11 nondiabetic control subjects were evaluated for gamma-interferon secretion in response to peptide stimulation in enzyme-linked immunospot assays. We identified peptides IAPP9-17, IGRP215-223, IGRP152-160, islet IA-2(172-180), and IA-2(482-490) as novel HLA-A*0201-restricted T-cell epitopes in type 1 diabetic patients. Interestingly, we observed a strong inverse correlation between the binding affinity of beta-cell peptides to HLA-A*0201 and CTL responses against those peptides in recent-onset type 1 diabetic patients. In addition, we found that self-reactive CTLs with specificity for an insulin peptide are frequently present in healthy individuals. These data suggest that many beta-cell epitopes are recognized by CTLs in recent-onset type 1 diabetic patients. These epitopes may be important in the pathogenesis of type 1 diabetes.

Nocardia Asteroides infection in a non-immunocompromised pediatric patient is extremely rare. We present a case of ethmoid sinusitis and orbital subperiosteal abscess caused by N. asteroides with a 20 year follow up and a review of the literature. N. asteroides was grown from intraoperative cultures for mycobacteria following surgical incision and drainage of the abscess. Postoperatively, the patient received a seven month course of trimethoprim-sulfamethozaxole and had no subsequent sequelae. Nocardia infections are common in immunocompromised patients. We present what we believe to be the first case of pediatric Nocardia sinusitis with 20-year follow up.

BACKGROUND:Bordetella pertussisinfections continue to be a major public health challenge in Canada. Polymerase chain reaction (PCR) assays to detectB pertussisare typically based on the multicopy insertion sequence IS481, which offers high sensitivity but lacks species specificity.METHODS: A novelB pertussisreal-time PCR assay based on the porin gene was tested in parallel with several previously published assays that target genes such as IS481,ptx-promoter, pertactin and a putative thialase. The assays were evaluated using a reference panel of common respiratory bacteria including differentBordetellaspecies and 107 clinical nasopharyngeal specimens. Discrepant results were confirmed by sequencing the PCR products.RESULTS: Analytical sensitivity was highest for the assay targeting the IS481element; however, the assay lacked specificity forB pertussisin the reference panel and in the clinical samples. False-positive results were also observed with assays targeting theptx-promoter and pertactin genes. A PCR assay based on the thialase gene was highly specific but failed to detect all reference strains ofB pertussis. However, a novel assay targeting the porin gene demonstrated high specificity forB pertussisboth in the reference panel and in clinical samples and, based on sequence-confirmed results, correctly predicted allB pertussis-positive cases in clinical samples. According to Probit regression analysis, the 95% detection limit of the new assay was 4 colony forming units/reaction.CONCLUSION: A novel porin assay forB pertussisdemonstrated superior performance and may be useful for improved molecular detection ofB pertussisin clinical specimens.

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-γ production induced by phorbol ester plus ionomycin, whereas it enhances IFN-γ production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.

X-linked lymphoproliferative disease (XLP) is a fatal immunological disorder that typically manifests following EBV infection. XLP patients exhibit a number of immune defects including abnormal T, B, and NK lymphocyte function. These defects have been attributed to mutations of Src homology 2 domain-containing gene 1A (SH2D1A), the gene encoding signaling lymphocytic activation molecule-associated protein (SAP), an intracellular adaptor molecule expressed in lymphocytes. We have observed that SAP knockout (SAPKO) mice and humans with XLP have a complete lack of CD1d-restricted NKT cells. As expected, SAPKO mice injected with the NKT cell agonist, α-galactosylceramide failed to generate NKT cell IFN-γ or IL-4. Furthermore, in contrast to wild-type littermates, SAPKO mice coinjected with OVA and α-galactosylceramide failed to mount OVA-specific CTL responses. These data suggest that an absence of NKT cells may underlie part of the immune dysregulation seen in SAPKO mice and in XLP patients.

2B4 is a SLAM-related receptor expressed on natural killer (NK) cells and cytotoxic T cells. It can regulate killing and gamma interferon secretion by NK cells, as well as T-cell-mediated cytotoxicity. There are conflicting data regarding the mechanism of action of 2B4. In these studies, we attempted to understand better the nature and basis of 2B4 signaling. Our studies showed that engagement of 2B4 on NK cells triggered a tyrosine phosphorylation signal implicating 2B4, Vav-1, and, to a lesser extent, SHIP-1 and c-Cbl. Structure-function analyses demonstrated that this response was defined by a series of tyrosine-based motifs in the cytoplasmic region of 2B4 and was not influenced by the extracellular or transmembrane segment of 2B4. In addition, the 2B4-induced signal was absolutely dependent on coexpression of SAP, a Src homology 2 (SH2) domain-containing adaptor associating with SLAM-related receptors and mutated in X-linked lymphoproliferative disease. It was also observed that 2B4 was detectably associated with the Src-related protein tyrosine kinase FynT in an immortalized NK cell line. Mutation of arginine 78 of SAP, a residue critical for binding of SAP to FynT, eliminated 2B4-mediated protein tyrosine phosphorylation, implying that SAP promotes 2B4 signaling most probably by recruiting FynT. Finally, despite the similarities in the signaling modalities of 2B4 and its relative SLAM, the natures of the tyrosine phosphorylation signals induced by these two receptors were found to be different. These differences were not caused by variations in the extent of binding to SAP but rather were dictated by the tyrosine-based sequences in the cytoplasmic domain of the receptors. Taken together, these data lead to a better understanding of 2B4 signaling. Furthermore, they provide firm evidence that the signals transduced by the various SLAM-related receptors are unique and that the specificity of these signals is defined by the distinctive arrays of intracytoplasmic tyrosines in the receptors.

Suspected colonies of methicillin-resistant Staphylococcus aureus (MRSA) on chromogenic, MRSASelect (BioRad) medium were confirmed using routine microbiological methods, and a multiplex real-time PCR (n = 108). Although the specificity of MRSASelect assessed at 24 h of incubation was much higher than that of 48 h (91.4 vs. 60 %), extending the incubation time to 48 h, along with PCR confirmation, increased the total number of true positive samples by 27.8 %. These results provide a cost effective method for sensitive and specific detection of MRSA.

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.

Activated T cells and their naive precursors display different functional avidities for peptide/MHC, but are thought to have identical antigenic repertoires. We show that, following activation with a cognate mimotope (NRP), diabetogenic CD8+ T cells expressing a single TCR (8.3) respond vigorously to numerous peptide analogs of NRP that were unable to elicit any responses from naive 8.3-CD8+ T cells, even at high concentrations. The NRP-reactive, in vivo activated CD8+ cells arising in pancreatic islets of nonobese diabetic mice are similarly promiscuous for peptide/MHC, and paradoxically this promiscuity expands as the aviditiy of the T cell population for NRP/MHC increases with age. Thus, activation and avidity maturation of T lymphocyte populations can lead to dramatic expansions in the range of peptides that elicit functional T cell responses.

X-linked lymphoproliferative disease (XLP) is characterized by a selective immune deficiency to EBV. The molecular basis of XLP has been attributed to mutations of signaling lymphocytic activation molecule-associated protein, an intracellular molecule known to associate with the lymphocyte-activating surface receptors SLAM and 2B4. We have identified a single nucleotide mutation in SLAM-associated protein that affects the NK cell function of males carrying the mutated gene. In contrast to normal controls, both NK and lymphokine-activated killer cell cytotoxicity was significantly reduced in two XLP patients. In addition to decreased baseline cytotoxicity, ligation of 2B4 significantly augmented NK lytic function in normal controls but failed to enhance the cytotoxicity of NK cells from XLP patients. These findings suggest that association of SAP with 2B4 is necessary for optimal NK/lymphokine-activated killer cytotoxicity and imply that alterations in SAP/2B4 signaling contribute to the immune dysfunction observed in XLP.

A vaccine against human immunodeficiency virus (HIV) is still awaited. Although the correlates of protection remain elusive, it is likely that CD8+ T cells play an important role in the control of this infection. To firmly establish the importance of these cells in protective immunity, a means of efficient elicitation of CD8+ T cell responses in the absence of antibody is needed and, when available, might represent a crucial step towards a protective vaccine. Here, a novel vaccine candidate was constructed as a multi-cytotoxic T lymphocyte (CTL) epitope gene delivered and expressed using modified vaccinia virus Ankara (MVA). The immunogen consists of 20 human, one murine and three rhesus macaque epitopes. The non-human epitopes were included so that the vaccine can be tested for immunogenicity and optimal vaccination doses, routes and regimes in experimental animals. Mice were immunized intravenously (i.v.) or intramuscularly (i.m.) using a single dose of 10(6) p.f.u. of the recombinant MVA and the induction of CTL was assessed. It was demonstrated that both administration routes induced specific CTL responses and that the i.v. route was moderately more immunogenic than the i.m. route. The frequencies of ex vivo splenocytes producing interferon-y upon MHC class I-restricted peptide stimulation were determined using an ELISPOT assay. Also, the correct processing and presentation of some HLA-restricted epitopes in human cells was confirmed.

Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four–amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4+ and CD8+ populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.

Summary The transport of calcium ions (Ca 2+ ) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca 2+ concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca 2+ "stores" and "store-operated" Ca 2+ entry via plasma membrane channels. However, the identity of molecular components conducting Ca 2+ currents within developing and mature T cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca 2+ channel Ca V 1.4 plays a cell-intrinsic role in the function, development, and survival of naive T cells. Plasma membrane Ca V 1.4 was found to be essential for modulation of intracellular Ca 2+ stores and T cell receptor (TCR)-induced rises in cytosolic-free Ca 2+ , impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) pathways. Collectively, these studies revealed that Ca V 1.4 functions in controlling naive T cell homeostasis and antigen-driven T cell immune responses.

Autoimmune diabetes in NOD mice can be prevented by application of Ags derived from Mycobacterium tuberculosis in the form of bacillus Calmette-Guérin or CFA. Disease protection by CFA is associated with a reduction in the numbers of pathogenic β-cell specific, self-reactive CTLs, a phenomenon dependent on the presence and function of NK cells. However, the mechanisms by which NK cells are activated and recruited by heat-killed M. tuberculosis within CFA are unclear. In this study, we report that CFA-mediated NK cell activation and mobilization is dependent on CD1d expression. The administration of M. tuberculosis from CFA results in rapid NKT cell activation and IFN-γ secretion both in vitro and in vivo. CFA-induced NKT cell activation is intact in MyD88−/− mice suggesting that the mechanism is independent of TLR signaling. Furthermore, CD1d expression was found to be essential for both M. tuberculosis-triggered NKT cell activation and CFA-mediated protection of NOD mice from diabetes. Collectively, these findings reveal hitherto previously unidentified roles for NKT cells in the adjuvant-promoting effects of CFA on innate and adaptive immunity.

OBJECTIVE Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children. RESEARCH DESIGN AND METHODS Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray. RESULTS NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I–related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase–AKT pathway. CONCLUSIONS These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.

CD4+FOXP3+ regulatory T cells are essential for immune tolerance, and murine studies suggest that their dysfunction can lead to type 1 diabetes (T1D). Human studies assessing regulatory T cell dysfunction in T1D have relied on analysis of FOXP3-expressing cells. Recently, distinct subsets of CD4+FOXP3+ T cells with differing function were identified. Notably, CD45RA−CD25intFOXP3low T cells lack suppressive function and secrete the proinflammatory cytokine IL-17. Therefore, we evaluated whether the relative fractions of CD4+FOXP3+ subsets are altered in new-onset T1D subjects. We report that children with new-onset T1D have an increased proportion of CD45RA−CD25intFOXP3low cells that are not suppressive and secrete significantly more IL-17 than other FOXP3+ subsets. Moreover, these T1D subjects had a higher proportion of both CD4+ and CD8+ T cells that secrete IL-17. The bias toward IL-17–secreting T cells in T1D suggests a role for this proinflammatory cytokine in the pathogenesis of disease.

Extravillous cytotrophoblast (EVT) migration, invasion and endovascular differentiation are regulated by a variety of growth factors, cytokines and adhesion molecules. Decidual natural killer cells (dNK) and their secreted cytokines probably modulate these processes. In this study, we used dNK-derived conditioned medium (dNK-CM) to investigate whether or not (i) dNK-CM was able to enhance capillary tube and network formation of an EVT cell line, HTR8/SVneo, on Matrigel, (ii) PI3K/AKT pathway and p38 MAPK pathway activation were involved, and (iii) HTR8/SVneo surface ICAM-1 played a role in the process of HTR8/SVneo endovascular differentiation. The results demonstrated that HTR8/SVneo constitutively form 'vascular' tubes and networks after culture on Matrigel. dNK-CM enhanced and maintained tube and network formation, acquiring an endothelium-like angiogenic morphology followed by increased VEGF-C production. HTR8/SVneo cell expression level of VE-cadherin, PECAM-1, VCAM-1 and alphavbeta3 was unaltered by dNK-CM, whereas ICAM-1 expression level was increased. Anti-human ICAM-1 blocking antibody inhibited HTR8/SVneo migration and partially reversed dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. PI3K/AKT and p38 MAPK pathways were activated in dNK-CM-mediated enhancement of HTR8/SVneo tube and network formation. The PI3K/AKT and p38 MAPK pathway inhibitors (LY294002 and SB202190, respectively) decreased dNK-CM-stimulated ICAM-1 induction, HTR8/SVneo migration, and reversed tube and network formation. The results suggest that dNK cell-secreted growth factors and cytokines participate in the regulation of HTR8/SVneo endothelium-like tube formation. Adhesion molecules, particularly ICAM-1, expressed on EVT may participate in the process. To our knowledge, this is the first report of a role for ICAM-1 in EVT angiogenesis, as previously reported for endothelial cells.

Autoimmune diabetes in nonobese diabetic (NOD) mice can be prevented by a single injection of complete Freund's adjuvant (CFA), but the mechanisms mediating protection remains unclear. We previously showed that NOD mice immunized with CFA have a markedly reduced incidence of diabetes that is associated with a significant decrease in the number of beta-cell-specific, autoreactive cytotoxic T lymphocytes and, furthermore, that the effect of CFA is mediated by natural killer (NK) cells. In this study, we report one mechanism by which NK cells regulate the onset of diabetes. Administration of CFA produced a rapid increase in NK cell frequency and function, including cytotoxicity and IFN-gamma secretion. By co-transferring NK cells from IFN-gamma-deficient (or wild-type) NOD mice and spleen cells from diabetic NOD mice to NOD/SCID recipients, we show that IFN-gamma secretion by NK cells significantly influences the effect of CFA protection. In contrast, NK cytotoxicity does not appear to participate in CFA-mediated protection from diabetes. Our findings demonstrate that NK cells mediate the protective effects of CFA through secretion of IFN-gamma.

Cytotoxic T-lymphocytes (CTLs) are considered to be essential for β-cell destruction in type 1 diabetes. However, few islet-associated peptides have been demonstrated to activate autoreactive CTLs from type 1 diabetic subjects. In an effort to identify novel epitopes, we used matrix-assisted algorithms to predict peptides of glial fibrillary acidic protein (GFAP), prepro-islet amyloid polypeptide (ppIAPP), and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP) that likely bind to HLA-A*0201 with a strong affinity and contain a COOH-terminal proteasomal cleavage site. Seven peptides stabilized HLA-A*0201 expression in binding assays and were used to stimulate peripheral blood mononuclear cells and were evaluated for granzyme B secretion. We found that 5 of 13 type 1 diabetic subjects and 4 of 6 antibody-positive relatives exhibited greater numbers of granzyme B–secreting cells in response to at least one putative epitope compared with healthy control subjects. The most prevalent responses in antibody-positive and type 1 diabetic subjects were to ppIAPP(9-17). Other peptides recognized by type 1 diabetic or antibody-positive subjects included GFAP(143-151), IGRP(152-160), and GFAP(214-222). These data implicate peptides of ppIAPP, GFAP, and IGRP as CTL epitopes for a heterogenous CD8+ T-cell response in type 1 subjects and antibody-positive relatives.

community has followed the period of excitement created by the identification of members of the chemokine receptor family as essential co-receptors for the en- try of HIV into CD4+T cells. This in- formation has now been absorbed and is prompting new insights into HIV pathogenesis and potential for new treatment. One key area of research has been to examine how expression of the principal co- receptors [CCR5 for macrophage- tropic (M-tropic), non-syncytium- inducing (NSI) virus; CXCR4 for T cell line-tropic, syncytium-inducing (SI) strains] might be regulated. At first, studies were limited to mRNA expression, but the use of an emerging armoury of specific mono- clonal antibodies, in combination with other T-cell markers, has

Allotransplantation of pancreatic islets represents a promising approach to treat type 1 diabetes. Destruction of beta-cells in islet allografts involves multiple immune mechanisms that lead to activation of caspases and apoptotic cell death. The X-linked inhibitor of apoptosis (XIAP) inhibits apoptosis induced by a variety of triggers, primarily by preventing the activation of caspases. To determine whether XIAP would protect beta-cells from apoptosis, we used a recombinant adenovirus to overexpress XIAP in transformed murine beta-cells and in freshly isolated islets. In vitro cytokine-induced beta-cell death was decreased to baseline levels in XIAP-transduced MIN-6 and NIT-1 cell lines compared with controls. To evaluate the potential of XIAP overexpression to prevent in vivo allogeneic graft rejection, we transduced Balb/c islets ex vivo with XIAP before transplantation into CBA mice with streptozotocin-induced diabetes. We observed that almost all mice receiving allografts of XIAP-expressing islets maintained normoglycemia until the experiment was terminated (45-72 days posttransplant), whereas control mice receiving islets transduced with adenovirus expressing LacZ were hyperglycemic by approximately 17 days posttransplantation due to graft rejection. Immunohistochemistry revealed preservation of beta-cells and clearance of infiltrating immune cells in the XIAP-expressing islet grafts. The in vitro allogeneic response of splenocytes isolated from recipients of XIAP-expressing grafts 8 weeks posttransplant was similar to that seen in nonprimed allogeneic mice, suggesting that XIAP overexpression may lead to the acceptance of islet allografts in diabetic recipients. Long-term protection of islet allografts by XIAP overexpression may enhance the survival of islet transplants in diabetes.

NK cells from individuals with X-linked lymphoproliferative (XLP) disease exhibit functional defects when stimulated through the NK receptor, 2B4 (CD244). These defects are likely a consequence of aberrant intracellular signaling initiated by mutations of the adaptor molecule SLAM-associated protein. In this report, we show that NK cells from individuals with XLP but not healthy individuals fail to phosphorylate and thereby inactivate glycogen synthase kinase-3 (GSK-3) following 2B4 stimulation. Lack of GSK-3 phosphorylation prevented the accumulation of the transcriptional coactivator β-catenin in the cytoplasm and its subsequent translocation to the nucleus. Potential signaling pathways leading from 2B4 stimulation to GSK-3 phosphorylation were also investigated. Ligation of 2B4 resulted in the phosphorylation of the guanine nucleotide exchange factor, Vav-1, and subsequent activation of the GTP-binding protein Rac-1 (but not Ras) and the serine-threonine kinase Raf-1 in healthy but not XLP-derived NK cells. In addition, the activity of MEK-2 (but not MEK-1) was up-regulated, and Erk1/2 was phosphorylated in normal NK cells but not those from an individual with XLP suggesting that these proteins relay SLAM-associated protein-dependent signals from 2B4. Finally, inactivation of GSK-3 using a specific inhibitor of GSK-3β increased the cytotoxicity and cytokine secretion of both healthy and XLP NK cells. These data indicate that the signaling of 2B4 in NK cells is mediated by GSK-3 and β-catenin, possibly through a signal transduction pathway that involves Vav-1, Rac-1, Raf-1, MEK-2, and Erk1/2 and that this pathway is aberrant in individuals with XLP.

Background. Neisseria meningitidis continues to be an important cause of invasive bacterial disease among children and young adults worldwide. In Canada, N. meningitidis strains that bear serogroups B and C polysaccharide capsules predominate. We report the first documented case of invasive meningococcal disease in an immunocompetent host caused by an acapsular strain of N. meningitidis containing the capsule null locus (cnl). Methods. Analysis of the isolate was performed with use of serological and molecular methods, including multilocus sequence typing and cnl gene identification. Analysis of 16S ribosomal RNA (rRNA) and porA genes was also performed to confirm the identity of the bacterium. Results. The patient was a healthy, immunocompetent 13-year-old child, and N. meningitidis was recovered from a sample of her cerebrospinal fluid before death. The isolate was nontypeable by both conventional antisera and indirect whole-cell enzyme-linked immuosorbent assay methods using antibodies to serogroups B, C, Y, and W135. The isolate was further identified as a cnl strain, serotype 15 (ST-198). N. meningitidis–specific DNA was identified in the isolate and in the pre- and postmortem specimens by 16S rRNA and porA gene analysis. Conclusions. This is the first reported case of fatal meningococcal disease caused by an acapsular cnl strain of N. meningitidis that was isolated from an immunocompetent host. Routine molecular diagnostic methods targeted at the cnl locus failed to detect this organism, indicating a need to determine the incidence of infection with cnl strains among patients with culture-negative invasive disease.

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.

A proportion of individuals vaccinated with live attenuated Oka varicella-zoster virus (VZV) vaccine subsequently develop attenuated chicken pox and/or herpes zoster. To determine whether postvaccination varicella infections are caused by vaccine or wild-type virus, a simple method for distinguishing the vaccine strain from wild-type virus is required. We have developed a TaqMan real-time PCR assay to detect and differentiate wild-type virus from Oka vaccine strains of VZV. The assay utilized two fluorogenic, minor groove binding probes targeted to a single nucleotide polymorphism in open reading frame 62 that distinguishes the Oka vaccine from wild-type strains. VZV DNA could be genotyped and quantified within minutes of thermocycling completion due to real-time monitoring of PCR product formation and allelic discrimination analysis. The allelic discrimination assay was performed in parallel with two standard PCR-restriction fragment length polymorphism (RFLP) methods on 136 clinical and laboratory VZV strains from Canada, Australia, and Japan. The TaqMan assay exhibited a genotyping accuracy of 100% and, when compared to both PCR-RFLP methods, was 100 times more sensitive. In addition, the method was technically simpler and more rapid. The TaqMan assay also allows for high-throughput genotyping, making it ideal for epidemiologic study of the live attenuated varicella vaccine.

The immune system of the skin has recently been exploited for the development of non-invasive vaccine technologies. However, one of the limitations of current vaccine protocols is the inefficient priming of cytotoxic T lymphocytes (CTL). In this study, we report that the application of either an immunodominant class I MHC restricted ovalbumin peptide or whole ovalbumin protein, to tape-stripped skin together with the co-application of the bacterial enterotoxin cholera toxin (CT) induces antigen-specific CTL. Tape-stripping (TS) was found to enhance the magnitude of antibody responses to co-administered protein and to promote the generation of antigen-specific IgG 2a responses. As well, both cholera toxin and tape-stripping enhanced epidermal dendritic cell (DC) immigration into draining lymph nodes. The adjuvant effect of co-administered cholera toxin and tape-stripping in promoting CTL priming was not dependent on IL-12. Epicutaneous immunization has previously been shown to induce robust antibody responses to administered protein antigen. We now demonstrate the induction of robust and persistent CTL responses to epicutaneously administered protein antigen. Epicutaneous immunization is cheap, simple and effective. These findings suggest the potential use of the skin for the generation of protective immune responses to both viral and tumor challenge.

Context.—Meningococcal infection has a high public profile because of its dramatic presentation, high fatality rate, and propensity to occur in outbreaks and clusters of cases. Use of a diagnostic polymerase chain reaction (PCR) assay could enhance laboratory confirmation of cases and guide the public health response in North America.Objective.—To assess the performance of a PCR assay for the diagnosis of meningococcal disease after its implementation in a North American setting and to evaluate sensitivity and specificity of the assay for the detection of prevalent bacterial isolates.Design.—Laboratory evaluation of the sensitivity and specificity of a PCR assay for Neisseria meningitidis and observational study of a series of cases comparing molecular diagnosis against the criterion standard of conventional laboratory diagnostic tests.Setting.—A Canadian province with a population of 4 million people.Patients.—Children and adults presenting with suspected meningococcal disease in British Columbia.Main Outcome Measures.—The sensitivity and specificity of the PCR assay when compared against standard laboratory methods.Results.—The PCR assay correctly identified all of 38 Canadian isolates of Neisseria meningitidis and correctly assigned the serogroup to each isolate. None of 57 other gram-positive or gram-negative bacteria or yeasts were detected by the PCR assay. In a clinical evaluation, for diagnosis of meningococcal disease, the PCR assay had a sensitivity and specificity of 91% and 76%, respectively, against conventional methods of diagnosis. Use of the PCR assay increased the laboratory confirmation of clinically suspected cases by 36%. During an outbreak, the PCR assay allowed serogroup determination in 3 of 7 cases, aiding in the public health decision to launch an immunization campaign.Conclusions.—The PCR assay is more sensitive than conventional methods for the diagnosis of meningococcal disease, and enhanced surveillance may help direct the public health response to the changing epidemiology of disease in North America.