CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo.

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

Anti-CTLA-4 treatment induces IL-10-producing ICOS+ regulatory T cells displaying IDO-dependent anti-inflammatory properties in a mouse model of colitis.

BACKGROUND AND AIMS: Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has been shown to act as a negative regulator of T cell function and has been implicated in the regulation of T helper 1 (Th1)/Th2 development and the function of regulatory T cells. Tests were carried out to determine whether anti-CTLA-4 treatment would alter the polarisation of naive T cells in vivo. METHODS: Mice were treated with anti-CTLA-4 monoclonal antibody (mAb) (UC10-4F10) at the time of immunisation or colonic instillation of trinitrobenzene sulfonic acid (TNBS). The cytokines produced by lymph node cells after in vitro antigenic stimulation and the role of indoleamine 2,3 dioxygenase (IDO) and of interleukin-10 (IL-10) were tested, and the survival of mice was monitored. RESULTS: Injection of anti-CTLA-4 mAb in mice during priming induced the development of adaptive CD4(+) regulatory T cells which expressed high levels of ICOS (inducible co-stimulator), secreted IL-4 and IL-10. This treatment inhibited Th1 memory responses in vivo and repressed experimental intestinal inflammation. The anti-CTLA-4-induced amelioration of disease correlated with IDO expression and infiltration of ICOS(high) Foxp3(+) T cells in the intestine, suggesting that anti-CTLA-4 acted indirectly through the development of regulatory T cells producing IL-10 and inducing IDO. CONCLUSIONS: These observations emphasise the synergy between IL-10 and IDO as anti-inflammatory agents and highlight anti-CTLA-4 treatment as a potential novel immunotherapeutic approach for inducing adaptive regulatory T cells.

An entirely cell-based system to generate single-chain antibodies against cell surface receptors.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

Similar T-cell immune responses induced by group M consensus env immunogens with wild-type or minimum consensus variable regions.

Consensus HIV-1 genes can decrease the genetic distances between candidate immunogens and field virus strains. To ensure the functionality and optimal presentation of immunologic epitopes, we generated two group-M consensus env genes that contain variable regions either from a wild-type B/C recombinant virus isolate (CON6) or minimal consensus elements (CON-S) in the V1, V2, V4, and V5 regions. C57BL/6 and BALB/c mice were primed twice with CON6, CON-S, and subtype control (92UG37_A and HXB2/Bal_B) DNA and boosted with recombinant vaccinia virus (rVV). Mean antibody titers against 92UG37_A, 89.6_B, 96ZM651_C, CON6, and CON-S Env protein were determined. Both CON6 and CON-S induced higher mean antibody titers against several of the proteins, as compared with the subtype controls. However, no significant differences were found in mean antibody titers in animals immunized with CON6 or CON-S. Cellular immune responses were measured by using five complete Env overlapping peptide sets: subtype A (92UG37_A), subtype B (MN_B, 89.6_B and SF162_B), and subtype C (Chn19_C). The intensity of the induced cellular responses was measured by using pooled Env peptides; T-cell epitopes were identified by using matrix peptide pools and individual peptides. No significant differences in T-cell immune-response intensities were noted between CON6 and CON-S immunized BALB/c and C57BL/6 mice. In BALB/c mice, 10 and eight nonoverlapping T-cell epitopes were identified in CON6 and CON-S, whereas eight epitopes were identified in 92UG37_A and HXB2/BAL_B. In C57BL/6 mice, nine and six nonoverlapping T-cell epitopes were identified after immunization with CON6 and CON-S, respectively, whereas only four and three were identified in 92UG37_A and HXB2/BAL_B, respectively. When combined together from both mouse strains, 18 epitopes were identified. The group M artificial consensus env genes, CON6 and CON-S, were equally immunogenic in breadth and intensity for inducing humoral and cellular immune responses.

The cytolytic molecules Fas ligand and TRAIL are required for murine thymic graft-versus-host disease.

Thymic graft-versus-host disease (tGVHD) can contribute to profound T cell deficiency and repertoire restriction after allogeneic BM transplantation (allo-BMT). However, the cellular mechanisms of tGVHD and interactions between donor alloreactive T cells and thymic tissues remain poorly defined. Using clinically relevant murine allo-BMT models, we show here that even minimal numbers of donor alloreactive T cells, which caused mild nonlethal systemic graft-versus-host disease, were sufficient to damage the thymus, delay T lineage reconstitution, and compromise donor peripheral T cell function. Furthermore, to mediate tGVHD, donor alloreactive T cells required trafficking molecules, including CCR9, L selectin, P selectin glycoprotein ligand-1, the integrin subunits alphaE and beta7, CCR2, and CXCR3, and costimulatory/inhibitory molecules, including Ox40 and carcinoembryonic antigen-associated cell adhesion molecule 1. We found that radiation in BMT conditioning regimens upregulated expression of the death receptors Fas and death receptor 5 (DR5) on thymic stromal cells (especially epithelium), while decreasing expression of the antiapoptotic regulator cellular caspase-8-like inhibitory protein. Donor alloreactive T cells used the cognate proteins FasL and TNF-related apoptosis-inducing ligand (TRAIL) (but not TNF or perforin) to mediate tGVHD, thereby damaging thymic stromal cells, cytoarchitecture, and function. Strategies that interfere with Fas/FasL and TRAIL/DR5 interactions may therefore represent a means to attenuate tGVHD and improve T cell reconstitution in allo-BMT recipients.

MRP3: a molecular target for human glioblastoma multiforme immunotherapy.

BACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.

Cryptococcus gattii VGIII isolates causing infections in HIV/AIDS patients in Southern California: identification of the local environmental source as arboreal.

Ongoing Cryptococcus gattii outbreaks in the Western United States and Canada illustrate the impact of environmental reservoirs and both clonal and recombining propagation in driving emergence and expansion of microbial pathogens. C. gattii comprises four distinct molecular types: VGI, VGII, VGIII, and VGIV, with no evidence of nuclear genetic exchange, indicating these represent distinct species. C. gattii VGII isolates are causing the Pacific Northwest outbreak, whereas VGIII isolates frequently infect HIV/AIDS patients in Southern California. VGI, VGII, and VGIII have been isolated from patients and animals in the Western US, suggesting these molecular types occur in the environment. However, only two environmental isolates of C. gattii have ever been reported from California: CBS7750 (VGII) and WM161 (VGIII). The incongruence of frequent clinical presence and uncommon environmental isolation suggests an unknown C. gattii reservoir in California. Here we report frequent isolation of C. gattii VGIII MATα and MATa isolates and infrequent isolation of VGI MATα from environmental sources in Southern California. VGIII isolates were obtained from soil debris associated with tree species not previously reported as hosts from sites near residences of infected patients. These isolates are fertile under laboratory conditions, produce abundant spores, and are part of both locally and more distantly recombining populations. MLST and whole genome sequence analysis provide compelling evidence that these environmental isolates are the source of human infections. Isolates displayed wide-ranging virulence in macrophage and animal models. When clinical and environmental isolates with indistinguishable MLST profiles were compared, environmental isolates were less virulent. Taken together, our studies reveal an environmental source and risk of C. gattii to HIV/AIDS patients with implications for the >1,000,000 cryptococcal infections occurring annually for which the causative isolate is rarely assigned species status. Thus, the C. gattii global health burden could be more substantial than currently appreciated.

Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation.

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.

Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

BACKGROUND: Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. METHODS: Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. RESULTS: The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. CONCLUSIONS: Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin.

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis

Background The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. Methods We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. Results We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. Conclusions JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis.

Intracellular antigenic changes associated with meiosis in the orthopteran Stauroderus scalaris

Monoclonal antibodies have been prepared against purified pachytene cells from grasshopper testes. Immunoblotting and immunofluorescence analyses identified those monoclonal antibodies which showed specificity for antigens in pachytene cells. Several antigenic changes were found to be associated with meiotic cells. Five monoclonal antibodies detected antigens which were located in the cytoplasm of premeiotic cells but were nuclear during meiosis. One monoclonal antibody showed a discrete cytoplasmic fluorescent pattern in meiotic, but not in premeiotic, cells. Another bound specifically to the nuclei of some epithelial cells at the base of follicles in mature testes.

Characteristics of a cetacean morbillivirus isolated from a porpoise (Phocoena phocoena)

A virus isolated from a porpoise during the 1988 seal epizootic was shown to be a morbillivirus. In order to determine the relationship of the virus to phocine distemper virus (PDV) a battery of monoclonal antibodies raised against canine distemper virus (CDV), PDV or the porpoise isolate were assessed for their ability to bind to CDV, PDV or porpoise virus epitopes in indirect immunofluorescence assays and ELISAs. The porpoise isolate contained several unique epitopes and several epitopes present on CDV and PDV were absent on the porpoise isolate. The data presented in this study indicate that the porpoise virus is an antigenically distinct morbillivirus and as such has been tentatively named as delphinoid distemper virus (DDV).

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

The pyrrolo-1,5-benzoxazepine, PBOX-6, inhibits the growth of breast cancer cells in vitro independent of estrogen receptor status and inhibits breast tumour growth in vivo

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.