Tatiana Goltsova

Adenovector-induced expression of human-CD40-ligand (hCD40L) by multiple myeloma cells: A model for immunotherapy

OBJECTIVE CD40L restores the antigen-presenting cell (APC) function of some B-cell tumors and induces professional APC maturation. We therefore evaluated the effects of transgenic CD40L expression on the behavior and immunogenicity of human multiple myeloma (MM) cells. MATERIALS AND METHODS CD40L expression was induced in a CD40(+) (RPMI 8226) and a CD40(-) (U266B1) human myeloma cell line (HMCL) by adenoviral vector gene transfer. The viability and proliferative activity of control HMCL and HMCL/CD40L were determined by daily trypan blue staining and methyl-3H-thymidine incorporation. Mixed lymphocyte reaction (MLR) with allogeneic mononuclear cells (MNCs) and coculture of allogeneic dendritic cells (DCs) with HMCL expressing transgenic CD40L were used to evaluate the APC function of modified HMCL as well as the role of bystander DCs in inducing an anti-tumor immune response. RESULTS CD40L expression significantly inhibited the growth of the CD40(+) HMCL and induced apoptosis. These effects were less evident for the CD40(-) HMCL. There was no upregulation of costimulatory molecules on either HMCL following CD40L expression. Both HMCL expressing transgenic CD40L induced maturation of bystander DCs and enhanced their ability to stimulate the proliferation of MNCs. DCs cultured with the poorly immunogenic RPMI 8226 expressing CD40L upregulated T-lymphocyte release of IFN-gamma and other Th1 cytokines (interleukin-2, tumor necrosis factor-alpha). CONCLUSIONS Our data suggest that transgenic expression of CD40L exerts a dual effect favoring generation of an immune response to human MM. Where the tumor cells are CD40(+), the engagement of CD40 antigen by CD40L on tumor cells induces their apoptosis, allowing uptake of tumor-associated antigen by professional APC. Independently of tumor-cell expression of CD40, transgenic expression of CD40L on tumor cells allows them to stimulate CD40(+) APC, to increase their maturation and their capacity to stimulate cytotoxic T lymphocytes (CTL) that recognize the tumor-derived antigens the APC may have engulfed.

Attraction and Activation of Dendritic Cells at the Site of Tumor Elicits Potent Antitumor Immunity

Tumor cells harbor unique genetic mutations, which lead to the generation of immunologically foreign antigenic peptide repertoire with the potential to induce individual tumor-specific immune responses. Here, we developed an in situ tumor vaccine with the ability to elicit antitumor immunity. This vaccine comprised an E1B-deleted oncolytic adenovirus expressing beta-defensin-2 (Ad-BD2-E1A) for releasing tumor antigens, recruiting and activating plasmacytoid dendritic cells (pDCs). Intratumoral injections of Ad-BD2-E1A vaccine inhibited primary breast tumor growth and blocked naturally occurring metastasis in mice. Ad-BD2-E1A vaccination induced potent tumor-specific T-cell responses. Splenic and intratumoral DCs isolated from Ad-BD2-E1A-immunized mice were able to stimulate or promote the differentiation of naive T cells into tumor-specific cytotoxic T cells. We further found that the increased numbers of mature CD45RA(+)CD8alpha(+)CD40(+) pDCs infiltrated into Ad-BD2-E1A-treated tumors. The antitumor effect of Ad-BD2-E1A vaccination was abrogated in toll-like receptor 4 (TLR4) deficient mice, suggesting the critical role of TLR4 in the induction of antitumor immunity by Ad-BD2-E1A. The results of this study indicate that in situ vaccination with the oncolytic BD2-expressing adenovirus preferentially attracts pDCs and promotes their maturation, and thus elicits potent tumor-specific immunity. This vaccine represents an attractive therapeutic strategy for the induction of individualized antitumor immunity.

Detection of Lymphoid and Myeloid Lineages in Infantile B-Cell Acute Lymphoblastic Leukemia With Mixed-Lineage Leukemia Rearrangement by Use of Flow Cytometry and Cytogenetic Analysis

Switch between lymphoid and myeloid cell lineage by a leukemic clone is an event with particularly poor prognostic significance. Lineage switch is not common and has previously been described by isolated case reports. Recently, a large, single-institution series drew attention to the association of this phenomenon with MLL (myeloid/lymphoid or mixed-lineage leukemia) abnormalities and highlighted the importance of differentiating between progression of original disease and evolution of treatment-associated malignancy. In this series, all patients who exhibited lineage switch died of disease, underlining the critical need for early recognition and stratification when making treatment decisions among this group. Flow cytometric monitoring has proven to be more sensitive and accurate than routine morphologic screening in evaluation for minimal residual disease and in tracking progression of leukemic clones. The present case illustrates how this methodology can augment detection and monitoring of leukemia that converts in lineage. This report emphasizes the importance of early recognition of the entity.