Bernadette Scott

Mouse Plasmacytoid Cells: Long-lived Cells, Heterogeneous in Surface Phenotype and Function, that Differentiate Into CD8+ Dendritic Cells Only after Microbial Stimulus

The CD45RAhiCD11cint plasmacytoid predendritic cells (p-preDCs) of mouse lymphoid organs, when stimulated in culture with CpG or influenza virus, produce large amounts of type I interferons and transform without division into CD8+CD205− DCs. P-preDCs express CIRE, the murine equivalent of DC-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN). P-preDCs are divisible by CD4 expression into two subgroups differing in turnover rate and in response to Staphylococcus aureus. The kinetics of bromodeoxyuridine labeling and the results of transfer to normal recipient mice indicate that CD4− p-preDCs are the immediate precursors of CD4+ p-preDCs. Similar experiments indicate that p-preDCs are normally long lived and are not the precursors of the short-lived steady-state conventional DCs. However, in line with the culture studies on transfer to influenza virus-stimulated mice the p-preDCs transform into CD8+CD205− DCs, distinct from conventional CD8+CD205+ DCs. Hence as well as activating preexistant DCs, microbial infection induces a wave of production of a new DC subtype. The functional implications of this shift in the DC network remain to be determined.

Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells.

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag “sequestration,” or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by ∼80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.

Tumor-Specific CD4+ T Cells Have a Major “Post-Licensing” Role in CTL Mediated Anti-Tumor Immunity

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4+ T cells synergize with CD8+ T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4+ T cells was not required for the generation of CTLs, because adoptive transfer of CD8+ T cells alone was sufficient, CD4+ T cells were required for the maintenance of CD8+ T cell numbers. Our data suggest that there is a correlation among the number of CD8+ T cells, in vivo CTL function, and IFN-γ production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4+ T cells are required for CD8+ T cell infiltration of the tumor.

Type I IFN Contributes to NK Cell Homeostasis, Activation, and Antitumor Function

This study demonstrates that type I IFNs are an early and critical regulator of NK cell numbers, activation, and antitumor activity. Using both IFNAR1- and IFNAR2-deficient mice, as well as an IFNAR1-blocking Ab, we demonstrate that endogenous type I IFN is critical for controlling NK cell-mediated antitumor responses in many experimental tumor models, including protection from methylcholanthrene-induced sarcomas, resistance to the NK cell-sensitive RMA-S tumor and cytokine immunotherapy of lung metastases. Protection from RMA-S afforded by endogenous type I IFN is more potent than that of other effector molecules such as IFN-γ, IL-12, IL-18, and perforin. Furthermore, cytokine immunotherapy using IL-12, IL-18, or IL-21 was effective in the absence of endogenous type I IFN, however the antimetastatic activity of IL-2 was abrogated in IFNAR-deficient mice, primarily due to a defect in IL-2-induced cytotoxic activity. This study demonstrates that endogenous type I IFN is a central mediator of NK cell antitumor responses.

Promyelocytic Leukemia Zinc Finger Protein Regulates Interferon-Mediated Innate Immunity

Summary Interferons (IFNs) direct innate and acquired immune responses and, accordingly, are used therapeutically to treat a number of diseases, yet the diverse effects they elicit are not fully understood. Here, we identified the promyelocytic leukemia zinc finger (PLZF) protein as a previously unrecognized component of the IFN response. IFN stimulated an association of PLZF with promyelocytic leukemia protein (PML) and histone deacetylase 1 (HDAC1) to induce a decisive subset of IFN-stimulated genes (ISGs). Consequently, PLZF-deficient mice had a specific ISG expression defect and as a result were more susceptible to viral infection. This susceptibility correlated with a marked decrease in the expression of the key antiviral mediators and an impaired IFN-mediated induction of natural killer cell function. These results provide new insights into the regulatory mechanisms of IFN signaling and the induction of innate antiviral immunity.

Cytokine gene therapy or infusion as treatment for solid human cancer

In the induction of tissue-directed immune responses, cytokines tend to be released within the affected tissues. We used two strategies to expose tumor tissues to continuous high levels of cytokines: First, a vaccinia interleukin (IL)2 recombinant was injected directly intratumorally 3-weekly at 10(7) pfus/dose in six patients with the solid tumor malignant mesothelioma (MM). No virus excretion was detectable. At each cycle vaccinia-IL-2 mRNA (SQ [semi-quantitative] reverse transcription polymerase chain reaction) was maximal 24-72 h following injection reduced at 8 days and faded by 21 days. No tumor regression occurred. Second, based on the success of granulocyte macrophage colony-stimulating factor (GM-CSF) in gene transfer experiments, we conducted a study using continuous intratumoral GM-CSF infusion in eight patients with MM using a portable pump at doses of 10 micro/cg/24 h over 8 weeks. Systemic neutrophil agglutination and local catheter-related difficulties occurred. Two patients demonstrated tumor necrosis, one of whom had a marked progressive mononuclear cell infiltration of the tumor associated with a partial response (>50% reduction in tumor area). Murine studies using our MM model in CBA and BALB/C mice have demonstrated that B7-1 and allo-class I transfections induce strong tumor-specific cytotoxic T lymphocyte responses: GM-CSF, IL-12, and IL-2 induced mixed nonspecific plus specific responses, whereas B7-2 and class II transfections were not effective. We conclude that increased intratumoral cytokine concentrations can be generated using both gene transfer and cytokine infusion approaches; however, both have their limitations and, at this stage, have not produced dramatic antitumor effects in humans.

Infiltration of a Mesothelioma by IFN-γ-Producing Cells and Tumor Rejection after Depletion of Regulatory T Cells

Depletion of CD4+CD25+Foxp3+ regulatory T cells (CD25+ Treg) with an anti-CD25 Ab results in immune-mediated rejection of tolerogenic solid tumors. In this study, we have examined the immune response to a mesothelioma tumor in mice after depletion of CD25+ cells to elucidate the cellular mechanisms of CD25+ Treg, a subject over which there is currently much conjecture. Tumor rejection was found to be primarily due to the action of CD8+ T cells, although CD4+ cells appeared to play some role. Depletion of CD25+ cells resulted in an accumulation in tumor tissue of CD4+ and CD8+ T cells and NK cells that were producing the potent antitumor cytokine IFN-γ. Invasion of tumors by CD8+ T cells was partially dependent on the presence of CD4+ T cells. Although a significant increase in the proliferation and number of tumor-specific CD8+ T cells was observed in lymph nodes draining the tumor of anti-CD25-treated mice, this effect was relatively modest compared with the large increase in IFN-γ-producing T cells found in tumor tissue, which suggests that the migration of T cells into tumor tissue may also have been altered. Depletion of CD25+ cells did not appear to modulate antitumor CTL activity on a per cell basis. Our data suggests that CD25+ Treg limit the accumulation of activated T cells producing IFN-γ in the tumor tissue and, to a lesser extent, activation and/or rate of mitosis of tumor-specific T cells in lymph nodes.

Factors determining the spontaneous activation of splenic dendritic cells in culture

Dendritic cells (DCs) serve as a link between the innate and adaptive immune systems. The activation state of DCs is crucial in this role. However, when DCs are isolated from lymphoid tissues, purified and placed in culture they undergo ‘spontaneous’ activation. The basis of this was explored, using up-regulation of DC surface MHC II, CD40, CD80 and CD86 as indicators of DC activation. No evidence was found for DC damage during isolation or for microbial products causing the activation. The culture activation of spleen DCs differed from that of Langerhans cells when released from E-cadherin-mediated adhesions, since E-cadherin was not detected and activation still occurred with β-catenin null DCs. Much of the activation could be attributed to DC—DC interactions. Although increases in surface MHC II levels occurred under all culture conditions tested, the increase in expression of CD40, CD80 and CD86 was much less under culture conditions where such interactions were minimised. DC-to-DC contact under the artificial conditions of high DC concentration in culture induced the production of soluble factors and these, in turn, induced the up-regulation of co-stimulatory molecules on the DC surface.

Upregulation of RCAN1 causes Down syndrome-like immune dysfunction

Background People with Down syndrome (DS) are more susceptible to infections and autoimmune disease, but the molecular genetic basis for these immune defects remains undetermined. In this study, we tested whether increased expression of the chromosome 21 gene RCAN1 contributes to immune dysregulation. Methods We investigated the immune phenotype of a mouse model that overexpresses RCAN1. RCAN1 transgenic (TG) mice exhibit T cell abnormalities that bear a striking similarity to the abnormalities described in individuals with DS. Results RCAN1-TG mice display T cell developmental defects in the thymus and peripheral immune tissues. Thymic cellularity is reduced by substantial losses of mature CD4 and CD8 thymocytes and medullary epithelium. In peripheral immune organs T lymphocytes are reduced in number and exhibit reduced proliferative capacity and aberrant cytokine production. These T cell defects are stem cell intrinsic in that transfer of wild type bone marrow into RCAN1-TG recipients restored medullary thymic epithelium and T cell numbers in the thymus, spleen and lymph nodes. However, bone marrow transplantation failed to improve T cell function, suggesting an additional role for RCAN1 in the non-haemopoietic compartment. Conclusions RCAN1 therefore facilitates T cell development and function, and when overexpressed, may contribute to immune dysfunction in DS.

Characterization of monoclonal antibodies specific to the transcription factor ETS-2 protein

ETS-2 is a member of the ETS family of transcription factors. ETS-2 was initially characterized as a nuclear oncogene and has been shown to play a role in regulation of apoptosis and cell cycle progression. Members of the ETS family display high sequence homology, thus, there is considerable controversy concerning the specificity of existing ETS-2 polyclonal antibodies that have been used to define ETS-2 function. We therefore embarked on the production of ETS-2 specific monoclonal antibodies. In this report, we describe the production and characterization of six antibodies and the localization of their target epitopes to distinct domains of the ETS-2 protein. Four antibodies are ETS-2 specific and two antibodies cross-react with ETS-1, an ETS family member with the highest amino acid sequence homology to ETS-2. This report provides a comprehensive evaluation of ETS-2 specific monoclonal antibodies verified using ETS-2 null cells. These antibodies can be used for EMSA, Western blotting, immunoprecipitation and immunofluorescence staining experiments. Collectively, these reagents are invaluable molecular tools that should help better understand the biological function of ETS-2.