Michael G. Tovey

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-α (TNF-α), IL-6, IL-12, and IFN-γ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-α/β. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Locally Administered TLR7 Agonists Drive Systemic Antitumor Immune Responses That Are Enhanced by Anti-CD40 Immunotherapy

Topical application of tumors with the TLR7 agonist imiquimod is an effective adjunct treatment for a range of primary dermatological cancers. However, for therapy to be effective against a broad range of solid tumor types, it must promote a strong systemic antitumor response that targets metastases in addition to primary tumor. We therefore investigated the potential of locally delivered imiquimod to stimulate an effective systemic antitumor response in a murine model of malignant mesothelioma (AB1-HA) with primary and distal tumors (dual tumor). Persistent delivery of imiquimod into primary tumor significantly retarded tumor growth in all treated mice compared with vehicle control. This local antitumor immune response required both CD8 T cells and NK cells, but not CD4 T cells, and was reliant on type I IFN induction. In vivo CTL studies and Ly6A/E staining of lymphocytes suggested that local imiquimod treatment had indeed induced a systemic, Ag-specific CD8 response. However, notably this response was not sufficient to retard the growth of an untreated distal tumor. Because local imiquimod treatment did not induce significant CD4 T cell responses, we investigated the efficacy of combining imiquimod with agonistic CD40 Ab (as a surrogate for CD4 T cell help). Combination of locally delivered imiquimod with systemic anti-CD40 immunotherapy not only significantly enhanced the local antitumor response, with 30% complete resolution, but it was also effective at significantly retarding growth of distal tumor. These results demonstrate that antitumor responses induced by locally delivered TLR7 agonists can be harnessed systemically for treating distal tumor.

Inhibition by Interferon of Thymidine Uptake in Chemostat Cultures of L1210 Cells

Mouse interferon preparations inhibited the incorporation of [3H]-thymidine into acid-insoluble material of mouse leukemia L1210 cells cultivated under steady-state conditions in a chemostat. Interferon exerted only a transitory and less pronounced effect on [3H]-deoxyadenosine incorporation and had no effect on the content of total DNA per cell. Study of [3H]-thymidine uptake at 1 degree into acid-soluble cellular material showed that interferon reduced the uptake of this labeled nucleoside whereas the uptake of [3H]-deoxyadenosine and [3H]-deoxy-D-glucose was not inhibited. The effect of interferon on [3H]-thymidine uptake occurred prior to the inhibitory action on cell multiplication. It is suggested that the inhibitory of [3H]-thymdine uptake reflected specific changes in the cell membrane of interferon-treated cells which may be relevant to the understanding of the antiviral and other biological effects of interferon.

Targeting the Effector Site with IFN-αβ-Inducing TLR Ligands Reactivates Tumor-Resident CD8 T Cell Responses to Eradicate Established Solid Tumors

Effective antitumor CD8 T cell responses may be activated by directly targeting the innate immune system within tumors. We investigated this response by injecting a range of TLR agonists into established tumors using a mouse model of malignant mesothelioma stably transduced with the hemagglutinin (HA) gene as a marker Ag (AB1-HA). Persistent delivery of the dsRNA mimetic poly(I:C) into established AB1-HA tumors resulted in complete tumor resolution in 40% of mice, with the remaining mice also showing a significant delay in tumor progression. Experiments in athymic nude mice along with CD8 depletion and IFN-αβ blocking studies revealed that tumor resolution required both CD8 T cells and type I IFN induction, and was associated with local changes in MHC class I expression. Surprisingly, however, tumor resolution was not associated with systemic dissemination or tumor infiltration of effector CD8 T cells. Instead, the antitumor response was critically dependent on the reactivation of tumor-resident CD8 T cell responses. These studies suggest that, once reactivated, pre-existing local CD8 T cell responses are sufficient to resolve established tumors and that in situ type I IFN is a determining factor.

Targeting Poly(I:C) to the TLR3-Independent Pathway Boosts Effector CD8 T Cell Differentiation through IFN-α/β

Poly(I:C) is an adjuvant used for antitumor treatment and vaccines because of its prominent effects on CD8 T cells and NK cells. Poly(I:C) binds TLR3 and this interaction is thought to be central for driving cell-mediated immune responses. We investigated the importance of TLR3 in poly(I:C)-mediated endogenous CD8 T cell responses using the pathogenic T cell stimulant Staphylococcus aureus enterotoxin A. While the responsive CD8 T cells expanded comparably in both wild-type and TLR3−/− mice, differentiation of effector CD8 T cells was enhanced by poly(I:C) in the TLR3−/− mice. A higher percentage of Ag-specific CD8 T cells became IFN-γ and TNF-α producers in the absence of TLR3 signaling. Consistent with this boosted response was the observation that TLR3-deficient cells synthesized less IL-10 compared with TLR3-sufficient cells in response to poly(I:C). Ultimately, however, the fundamental mechanism of CD8 effector T cell differentiation through the TLR3-independent pathway was shown to be completely IFN-α/β-dependent. Administration of IFN-α/β-neutralizing Abs abolished the poly(I:C) effects in TLR3−/− mice. These findings reveal specific roles of how dsRNA receptors shape CD8 T cell responses, which should be considered as poly(I:C) is authenticated as a therapeutic adjuvant used in vaccines.

Characteristics of the chemostat culture of murine leukemia L 1210 cells

Abstract Mouse leukemia L 1210 cells were cultivated under glucose limitation in a chemostat. More than 20 steady-states were established over 9 different dilution rates ranging from 0.20 day −1 (cell doubling time 83 h) to 2.0 days −1 (cell doubling time 8.3 h). The steady-states were characterized by: a constant cell number, constant cell volume, constant concentrations of DNA, RNA, and L-lactate (in the culture supernatant), a constant percentage of cells labelled by autoradiography, and constant rate of incorporation of [ 3 H]TdR, [ 3 H]uridine, and 14 C-labelled amino acids into cellular acid-precipitable material. Individual steady-states were maintained for periods up to 600 h continuous operation of the chemostat. A maximum output of 66.4 × 10 6 cells/h was obtained at a dilution rate of 1.3 day −1 . The glucose substrate constant was determined as 0.0063 mg/ml. The relationships between dilution rate and the steady-state cell concentration, glucose concentration, and output of L 1210 cells from the chemostat, were in general agreement with the theoretical curves. It was found that the principles of continuous culture derived from the study of microorganisms are to a large extent applicable to the cultivation of animal cells.

Interaction of the nuclear protein CBF1 with the kappaB site of the IL-6 gene promoter.

The nuclear protein CBF1 has been shown to function as an intermediate to target transcription factors,such as the activated Notch receptor,to specific DNA sites. In this paper,we show that CBF1 from cell lines of different origin is able to bind to the[kappa]B site of the IL-6 promoter. By transfection analyses performed in HeLa cells,we demonstrate that overexpressed CBF1 acts as a negative regulator of IL-6 gene transcription and is unable to elicit Notch-dependent activation of this gene. Analyses of protein-DNA interactions indicate that the topology of the complex formed by CBF1 and the target DNA is subtly affected by sequencessurrounding the recognition site. Furthermore,we show that CBF1 induces DNA bending. This finding suggests that CBF1 may influence IL-6 gene transcription by determining a specific conformation of the promoter region.

Constitutive expression of specific interferon isotypes in peripheral blood leukocytes from normal individuals and in promonocytic U937 cells

Constitutive expression of IFN‐α5 and IFN‐β was detected in different lymphoid cells including peripheral blood mononuclear cells from normal individuals following amplification of IFN mRNA by reverse transcriptase–polymerase chain reaction and direct sequencing of the amplified product. The activated form of the interferon‐induced transcription factor complex ISGF3 was also dectected in nuclear extracts from uninduced cells. Culture supernatants from uninduced U937 cells were also found to activate an ISRE cloned upstream of the luciferase reporter gene, indicating the presence of endogenous IFN activity equivalent to approximately 0.3 to 0.5 IU/mL. This endogenous IFN was also shown to play a role in mamtaining the basal level of expression of the major histocompatibility class I genes in lymphoid cells. These results suggest that IFN‐α5 and IFN‐β are produced at low levels in normal tissues and play an important role in the regulation of cell function and in the maintenance of homeostasis.

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

Early effect of interferon on mouse leukemia cells cultivated in a chemostat.

Mouse interferon preparations inhibited the multiplication of mouse leukemia L 1210 cells cultivated under steady-state conditions in a chemostat. The use of this sensitive and controlled system led to the detection of a rapid inhibition in the incorporation of (3-H)thymidine into cellular acid-precipitable material 2 hr after the addition of interferon, whereas an effect on cell multiplication was not detected until 22 hr later. Interferon exerted only a transitory effect on the incorporation of (3-H)uridine into acid-precipitable material and no effect on the incorporation of 14-C-amino acids into cellular protein. It is suggested that the chemostat offers many advantages for the investigation of those physiologic factors or chemotherapeutic substances that modify cell division.