John T. Kung

Multiple Lineages of Human Breast Cancer Stem/Progenitor Cells Identified by Profiling with Stem Cell Markers

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44+/CD24-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR+/ESA+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44+/CD24−/low, ESA+, CD133+, CXCR4+ and PROCR+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

Both Virus and Tumor Necrosis Factor Alpha Are Critical for Endothelium Damage in a Mouse Model of Dengue Virus-Induced Hemorrhage

ABSTRACT Hemorrhage is a common clinical manifestation in dengue patients. However, the pathogenic mechanism of dengue virus (DV)-induced hemorrhage awaits clarification. We established a mouse model of DV hemorrhage using immunocompetent C57BL/6 mice by injecting DV serotype 2 strain 16681 intradermally. While inoculation of 3 × 109 PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, one out of three of those injected with 4 × 107 to 8 × 107 PFU developed hemorrhage in the subcutaneous tissues. The mice that were inoculated with 4 × 107 to 8 × 107 PFU but that did not develop hemorrhage were used as a basis for comparison to explore the pathogenic mechanism of dengue hemorrhage. The results showed that mice with severe thrombocytopenia manifested signs of vascular leakage and hemorrhage. We observed that high viral titer, macrophage infiltration, and tumor necrosis factor alpha (TNF-α) production in the local tissues are three important events that lead to hemorrhage. Immunofluorescence staining revealed that DV targeted both endothelial cells and macrophages. In addition, the production of high levels of TNF-α in tissues correlated with endothelial cell apoptosis and hemorrhage. By comparing TNF-α−/− to IgH−/−, C5−/−, and wild-type mice, we found that TNF-α was important for the development of hemorrhage. In vitro studies showed that mouse primary microvascular endothelial cells were susceptible to DV but that TNF-α enhanced DV-induced apoptosis. Our mouse model illustrated that intradermal inoculation of high titers of DV predisposes endothelial cells to be susceptible to TNF-α-induced cell death, which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of the innate immune response to dengue hemorrhage.

Recombinant Adeno-Associated Virus Expressing Human Papillomavirus Type 16 E7 Peptide DNA Fused with Heat Shock Protein DNA as a Potential Vaccine for Cervical Cancer

ABSTRACT In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242–2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine.

Induction of CD8 T Cells by Vaccination with Recombinant Adenovirus Expressing Human Papillomavirus Type 16 E5 Gene Reduces Tumor Growth

ABSTRACT The potential of the E5 protein as a tumor vaccine candidate has not been explored yet. In this study, we evaluate the human papillomavirus type 16 (HPV-16) E5 protein delivered by an adenovirus vector as a tumor vaccine for cervical lesions. The results demonstrate that a single intramuscular injection of a recombinant adenovirus carrying the HPV-16 E5 gene into syngeneic animals can reduce the growth of tumors which contain E5 gene expression. Moreover, the E5 vaccine-induced tumor protection occurs through CD8 T cells but not through CD4 T cells in in vitro assays. In addition, our studies using knockout mice with distinct T-cell deficiencies confirm that cytotoxic T-lymphocyte-induced tumor protection is CD8 dependent but CD4 independent. Hence, HPV-16 E5 can be regarded as a tumor rejection antigen.

Marginal Zone B Cell Is a Major Source of Il-10 in Listeria monocytogenes Susceptibility

Rag-1–knockout (KO) mice are highly resistant to Listeria monocytogenes infection. The role played by the many Rag-1–dependent lymphocyte lineages was studied using a genetic approach in which each Rag-1–dependent lymphocyte lineage was eliminated one at a time. Only B cell-deficient Igh-KO mice displayed reduced bacterial load and improved survival upon Listeria infection. Listeria infection of Rag-1–KO and Il-10–KO hosts that had been adoptively transferred with wild-type marginal zone B (MZB) cells, but not follicular B cells, resulted in heightened bacterial load and increased Il-10 production in the spleen, but not the liver. This MZB cell-dependent increase in bacterial load was eliminated by anti–Il-10 mAb. In addition, Listeria infection of MZB cell-deficient Rbpj-cKO mice showed decreased bacterial load and increased survival. Whereas multiple cell types have been shown to be capable of Il-10 production, our results indicate that the MZB cell is the most dominant and relevant Il-10 source in the context of Listeria susceptibility. In marked contrast to the generally protective nature of MZB cells in defending against pathogenic infection, our results demonstrate that MZB cells play a detrimental role in Listeria infection and possibly other infections as well.

Immunization with Apoptotic Phagocytes Containing Histoplasma capsulatum Activates Functional CD8+ T Cells To Protect against Histoplasmosis

ABSTRACT We have previously revealed the protective role of CD8+ T cells in host defense against Histoplasma capsulatum in animals with CD4+ T cell deficiency and demonstrated that sensitized CD8+ T cells are restimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated functional CD8+ T cells whose contribution was equal to that of CD4+ T cells in protection against Histoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8+ T cell but not the CD4+ T cell response to pulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whose CD8+ T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8+ T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected host is associated with CD8+ T cell activation and that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently evokes a protective CD8+ T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8+ T cell as well as CD4+ T cell responses to Histoplasma infection.

The roles of interleukin-1 and interleukin-1 receptor antagonist in antigen-specific immune responses

Despite evidence that interleukin (IL)-1 promotes the proliferation of some T helper 2 (Th2) cell clones in vitro, the physiological role of IL-1 in the regulation of antigen-specific immune responses remains undefined. Using a liposome-DNA delivery system, we transiently expressed IL-1 receptor antagonist (IL-1Ra) to suppress IL-1 functions at the site of the antigen-specific primary immune response. Our data indicate, for the first time, that IL-1Ra downregulates antigen-specific IL-4 and IgE responses, with concomitant enhancement of interferon-gamma and IgG2a responses in vivo. In addition, IL-1 can promote Th2 development in an IL-4-independent manner in vitro. Thus, the balance between endogenous IL-1 and IL-1Ra during the primary immune response can be an important factor in determining the antigen-specific effector function of T cells.

Immunodeficient mouse models with different disease profiles by in vivo infection with the same clinical isolate of enterovirus 71.

ABSTRACT Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia-Pacific region. Disease manifestations include subclinical infection, common-cold-like syndromes, hand-foot-and-mouth disease (HFMD), uncomplicated brain stem encephalitis, severe dysregulation of the autonomic nerve system, fatal pulmonary edema, and cardiopulmonary collapse. To date, no effective vaccine or treatment is available. A user-friendly and widely accessible animal model for researching EV71 infection and pathogenesis is urgently needed by the global community, both in academia and in industry.

Rapid and Selective Expansion of Nonclonotypic T Cells in Regulatory T Cell-Deficient, Foreign Antigen-Specific TCR-Transgenic Scurfy Mice: Antigen-Dependent Expansion and TCR Analysis

Foreign Ag-specific TCR-transgenic (Tg) mice contain a small fraction of T cells bearing the endogenous Vβ and Vα chains as well as a population expressing an intermediate level of Tg TCR. Importantly, these minor nonclonotypic populations contain ≥99% of the CD4+Foxp3+ regulatory T cells (Treg) and, despite low overall Treg expression, peripheral tolerance is maintained. In the OT-II TCR (OVA-specific, Vβ5highVα2high) Tg scurfy (Sf) mice (OT-II Sf) that lack Treg, nonclonotypic T cells markedly expanded in the periphery but not in the thymus. Expanded T cells expressed memory/effector phenotype and were enriched in blood and inflamed lungs. In contrast, Vβ5highVα2high clonotypic T cells were not expanded, displayed the naive phenotype, and found mainly in the lymph nodes. Importantly, Vβ5neg T cells were able to transfer multiorgan inflammation in Rag1−/− recipients. T cells bearing dual TCR (dual Vβ or dual Vα) were demonstrated frequently in the Vβ5int and Vα2int populations. Our study demonstrated that in the absence of Treg, the lack of peripheral expansion of clonotypic T cells is due to the absence of its high-affinity Ag OVA. Thus, the rapid expansion of nonclonotypic T cells in OT-II Sf mice must require Ag (self and foreign) with sufficient affinity. Our study has implications with respect to the roles of Ag and dual TCR in the selection and regulation of Treg and Treg-controlled Ag-dependent T cell expansion in TCR Tg and TCR Tg Sf mice, respectively.

Pervasive and stochastic changes in the TCR repertoire of regulatory T-cell-deficient mice

We hypothesize that regulatory T-cell (Treg)-deficient strains have an altered TCR repertoire in part due to the expansion of autoimmune repertoire by self-antigen. We compared the Vbeta family expression profile between B6 and Treg-lacking B6.Cg-Foxp3(sf)(/Y) (B6.sf) mice using fluorescent anti-Vbeta mAbs and observed no changes. However, while the spectratypes of 20 Vbeta families among B6 mice were highly similar, the Vbeta family spectratypes of B6.sf mice were remarkably different from B6 mice and from each other. Significant spectratype changes in many Vbeta families were also observed in Treg-deficient IL-2 knockout (KO) and IL-2Ralpha KO mice. Such changes were not observed with anti-CD3 mAb-treated B6 mice or B6 CD4+CD25- T cells. TCR transgenic (OT-II.sf) mice displayed dramatic reduction of clonotypic TCR with concomitant increase in T cells bearing non-transgenic Vbeta and Valpha families, including T cells with dual receptors expressing reduced levels of transgenic Valpha and endogenous Valpha. Collectively, the data demonstrate that Treg deficiency allows polyclonal expansion of T cells in a stochastic manner, resulting in widespread changes in the TCR repertoire.