Zhuang Chen

Efficient antitumor immunity derived from maturation of dendritic cells that had phagocytosed apoptotic/necrotic tumor cells

Dendritic cells (DCs) that acquired antigen from apoptotic tumor cells are able to induce major histocompatibility complex (MHC) class I‐restricted cytotoxic T lymphocytes and antitumor immunity. In the present study, we investigated the efficiency of antitumor immunity derived from DCs that had phagocytosed apoptotic/necrotic BL6‐10 melanoma cells compared with that of DCs pulsed with the tumor mTRP2 peptide. Our data showed that phagocytosis of apoptotic/necrotic tumor cells resulted in maturation of DCs with up‐regulated expression of proinflammatory cytokines [interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α, interferon‐γ and granulocyte‐macrophage colony‐stimulating factor], chemokines (MIP‐1α, MIP‐1β and MIP‐2), the CC chemokine receptor CCR7 and the cell surface molecules (MHC class II, CD11b, CD40 and CD86), and down‐regulated expression of the CC chemokine receptors CCR2 and CCR5. These mature DCs displayed enhanced migration toward the CC chemokine MIP‐3β in a chemotaxis assay in vitro and to the regional lymph nodes in an animal model in vivo. Our data also showed that vaccination with DCs that had phagocytosed apoptotic/necrotic BL6‐10 cells was able to (i) more strongly stimulate allogeneic T‐cell proliferation in vitro, (ii) induce an in vivo Th1‐type immune response leading to more efficient tumor‐specific cytotoxic CD8+ T‐cell‐mediated immunity and (iii) eradicate lung metastases in all 6 vaccinated mice compared with mice vaccinated with DCs pulsed with the tumor mTRP2 peptide, in which lung metastases were reduced (mean number of 16 per mouse) but not completely eradicated. Therefore, DCs that had phagocytosed apoptotic/necrotic tumor cells appear to offer new strategies in DC cancer vaccines.

Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8 + cytotoxic T-cell activation and antitumor immunity

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1β and IL-12), chemokines (RANTES, IP-10, and MIP-1α), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DCCD40L) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8+ cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus–transfected DC (DCpLpA) could only protect mice from challenge of a low dose (0.5×105 cells per mouse, 8/8 mice), but not a high dose (3×105 cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L–transfected DCCD40L induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.

Tumour necrosis factor-α (TNF-α) transgene-expressing dendritic cells (DCs) undergo augmented cellular maturation and induce more robust T-cell activation and anti-tumour immunity than DCs generated in recombinant TNF-α

Tumour antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of anti‐tumour immune responses. It has been previously reported that tumour necrosis factor‐α (TNF‐α) is required for DC activation and subsequent induction of optimal immune responses, and thus DCs for anti‐tumour vaccination are often generated by culture in exogenous TNF‐α. In the present study, we investigated the effect on anti‐tumour immunity of vaccination with Mut1 tumour peptide‐pulsed DCs engineered to express a TNF‐α transgene. Our data shows that transfection of DCs with recombinant adenovirus AdV‐TNF‐α resulted in greater maturation of the DCs than occurred with control DCs cultured in exogenous TNF‐α, as determined by up‐regulated expression of pro‐inflammatory cytokines (e.g. interleukins 1β and 18), chemokines [e.g. interferon‐γ‐inducible protein‐10 and macrophage inflammatory protein‐1β (MIP‐1β)], the CC chemokine receptor CCR7, and immunologically important cell surface molecules (CD40, CD86 and intercellular adhesion molecule‐1). These transgenic DCs stimulated stronger allogeneic T‐cell responses in vitro and T‐cell activation in vivo; displayed 2·4‐fold enhanced chemotactic responses to the MIP‐3βin vitro (P<0·05); and, perhaps most importantly, trafficked into the draining lymph nodes dramatically (seven‐fold, P<0·01) more efficiently than the control DCs. Our data also demonstrate that vaccination of mice with Mut1 peptide‐pulsed, AdV‐TNF‐α‐transfected DCs stimulated more efficient in vitro Mut1‐specific CD8+ cytotoxic T‐cell responses and solid tumour immunity in vivo, when compared to the in vitro TNF‐α‐cultivated DCs. Thus, DCs engineered to secrete TNF‐α may offer a new strategy in DC cancer vaccines.

DNA microarray analysis of the gene expression profiles of naı̈ve versus activated tumor-specific T cells

T cells are a key element in effective cancer immunity, recognizing MHC-antigen peptide complexes on the surface of antigen presenting cells and translating these signals into cytotoxic effector T cell responses. In this study, we systematically investigated by DNA array analysis the expression profiles of 514 immunologically relevant genes in naïve and SP2/0 tumor-specific activated mouse T cell populations. Our data shows that naïve T cells expressed 37 (i.e., 7.6% of the 514) transcripts with expression level (EL) values of > or =2.0, while the activated T cells expressed 101 such transcripts. The expression levels of 9 (1.75% of 514) of the shared transcripts were equivalent in the two populations of T cells. Ninety-six genes were differently expressed upon T cell activation, with 71 (13.81%) being up-regulated and 25 (4.86%) down-regulated. The list of significantly affected genes includes numerous cytokines and their receptors (e.g., IL-2Ralpha, IL-6Ralpha, IL-7Ralpha, IL-16, IL-17R, TGF-beta), chemokines and chemokine receptors (e.g., RANTES, CCR7, CXCR4), alternate surface proteins (e.g., 4-1BB, GITR, integrins-alphaL and -beta7, L-selectin, CD6, CD45 and EMMPRIN), cytoplasmic signaling intermediates (e,g., GATA-3, 14-3-3-eta, CIS1, SMAD4 and JAK1) and an array of other molecules (e.g., NFkappa-B inducing kinase, LTBP3 and persephin), several of which are associated with Th1 responses, and T cell self-regulation or migration. Taken together, our data contribute to our understanding of the generalized processes that accompany T cell activation and, more specifically, to our understanding of the processes associated with T cell activation during antitumor responses.

Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor

The present study uses an in vivo murine tumor model expressing the human HER-2/neu antigen to evaluate the potential vaccine using dendritic cells (DCs) infected with adenovirus AdVHER-2. We first investigated whether infected DCs (DCHER-2) engineered to express HER-2/neu could induce HER-2/neu-specific immune responses. Our data showed that (i) AdVHER2-infected DCHER-2 expressed HER-2/neu by Western blot and flow cytometric analysis, and (ii) vaccination of mice with DCHER-2 induced HER-2/neu-specific cytotoxic T-lymphocyte (CTL) responses, but protected only 25% of vaccinated mice from challenge of 3×105 MCA26/HER-2 tumor cells. Further, to enhance the efficacy of DCHER-2 vaccine, we coinfected DCs with both AdVHER-2 and AdVTNF-α. The infected DCs (DCHER-2/TNF-α) displayed the expression of both HER-2/neu and TNF-α by flow cytometric and ELISA analysis. We next investigated whether DCHER-2/TNF-α could induce stronger HER-2/neu-specific immune responses. We found that DCHER-2/TNF-α displayed up-regulation of immunologically important CD40, CD86, and ICAM-I molecules compared with DCHER-2, indicating that the former ones are more mature forms of DCs. Vaccination of DCHER-2/TNF-α induced stronger allogeneic T-cell proliferation and 36% enhanced HER-2/neu-specific T-cell responses in vitro than DCHER-2 cells. More importantly, it stimulated the significant anti–HER-2/neu immunity in vivo, which protected 8/8 mice from challenge of 3×105 MCA26/HER-2 tumor cells. Therefore, DCs genetically engineered to express both the tumor antigen and cytokines such as TNF-α as an immunoadjuvant are likely to represent a new direction in DC vaccine of cancer.

Combined radiation therapy and dendritic cell vaccine for treating solid tumors with liver micro‐metastasis

Tumor metastasis and relapse are major obstacles in combating human malignant diseases. Neither radiotherapy alone nor injection of dendritic cells (DCs) can successfully overcome this problem. Radiation induces tumor cell apoptosis and necrosis, resulting in the release of tumor antigen and danger signals, which are favorable for DC capturing antigens and maturation. Hence, the strategy of combined irradiation and DC vaccine may be a novel approach for treating human malignancies and early metastasis.

Dendritic cells engineered to express the Flt3 ligand stimulate type I immune response, and induce enhanced cytoxic T and natural killer cell cytotoxicities and antitumor immunity

Tumor antigen presentation by dendritic cells (DCs) to T cells in lymphoid organs is crucial for induction of antitumor immune responses. Fms‐like tyrosine kinase 3 ligand (Flt3L) is a regulator of hematopoietic cell development.

Regression of engineered myeloma cells secreting interferon-γ-inducing factor is mediated by both CD4+/CD8+ T and natural killer cells

IL-18 is a novel cytokine that stimulates T and NK cell activity and has potent antitumor effects. In this study, a mouse IL-18 gene was transfected into the mouse myeloma cell line J558. Our data demonstrated that (i) inoculation of 0.5x10(6) engineered tumor cells J558/IL-18 into syngeneic mice induced a Th1 dominant immune response and resulted in tumor regression in all 8/8 mice; (ii) the IL-18 antitumor effect was significantly decreased in mice depleted of either the CD4(+), or CD8(+), or NK cell subset, respectively but was completely abrogated in mice depleted of both CD4(+) and CD8(+) T cells; (iii) in vivo neutralization of IFN-gamma was accompanied by the growth of J558/IL-18 tumor in all the mice; and (iv) the J558/IL-18 tumor regression further induced protective immunity against a subsequent challenge by the parental J558 tumor, which is mediated by CD8(+) T cells as examined in the cytotoxicity assay in vitro and in the animal study in vivo. Taken together, our findings indicate that: (i) IL-18 can induce antitumor immune responses mediated by both CD4(+)/CD8(+) T cells and NK cells; and (ii) it is associated with IFN-gamma production. This study thus highlights the potential utility of IL-18 as an antitumor agent, a role that it can fulfil alone or in combination with other immunomodulatory cytokines such as IL-12.