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Abstract Background The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. Results In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRαβ+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vβ repertoire. Conclusions Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.

Microphthalmia-associated transcription factor ( MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth–associated molecules may be independent of MITF and dependent on BRAFV600E. Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAFV600E inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAFV600E significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment. ( Cancer Sci 2009; 100: 1863–1869) [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) play a key role in innate and adaptive immunity but the access to sufficient amount of DCs for basic and translational research has been limited. We established a novel ex vivo system to develop and expand DCs from hematopoietic stem/progenitor cells (HPCs). Both human and mouse HPCs were expanded first in feeder culture supplemented with c-Kit ligand (KL, stem cell factor, steel factor or CD117 ligand), Flt3 ligand (fms-like tyrosine kinase 3, Flt3L, FL), thrombopoietin (TPO), IL-3, IL-6, and basic fibroblast growth factor (bFGF), and then in a second feeder culture ectopically expressing all above growth factors plus GM-CSF and IL-15. In the dual culture system, CD34+ HPCs differentiated toward DC progenitors (DCPs), which expanded more than five orders of magnitude. The DCPs showed myeloid DC surface phenotype with up-regulation of transcription factors PU.1 and Id2, and DC-related factors homeostatic chemokine ligand 17 (CCL17) and beta-chemokine receptor 6 (CCR6). Multiplex ELISA array and cDNA microarray analyses revealed that the DCPs shared some features of IL-4 and IL-15 DCs but displayed a pronounced proinflammatory phenotype. DCP-derived DCs showed antigen-uptake and immune activation functions analogous to that of the peripheral blood-derived DCs. Furthermore, bone marrow HPC-derived DCP vaccines of tumor-bearing mice suppressed tumor growth in vivo. This novel approach of generating DCP-DCs, which are different from known IL-4 and IL-15 DCs, overcomes both quantitative and qualitative limitations in obtaining functional autologous DCs from a small number of HPCs with great translational potential. [ABSTRACT FROM AUTHOR]

MITF (microphthalmia-associated transcription factor) is a family of transcriptional factors controlling melanocyte differentiation. However, the roles of MITF on the cell proliferation have been controversial. Previous works have suggested that MITF negatively regulates cell proliferation or apoptosis in melanocytes, which is mediated by induction of p16INK4a, p21 or Bcl-2. But a recent study showed gene amplification of MITF in melanoma tissues (10-21%) associated with increase of the MITF protein. Furthermore, MITF gene amplification was a prognostic factor for survival, and several in vitro studies suggested that the MITF protein is attributed to increased cell proliferation, suggesting that MITF is an oncogene. Presently, the role of MITF in melanoma and melanomagenesis is not clear. We have attempted to investigate the role of MITF in malignant phenotypes of melanoma cells by using HIV-mediated RNA interference (RNAi) in a panel of melanoma cell lines with different status of the MITF expression to clarify the significance of MITF as a target for gene therapy, and to find any factors affecting sensitivity for the MITF inactivation. Furthermore, we have attempted to evaluate any additional effects by simultaneous suppression of both MITF and BRAFV600E on the melanoma growth, since BRAFV600E, the most frequent form of human BRAF mutation, is attributed to the enhanced MAPK signaling and several malignant phenotypes of melanoma.We have constructed two short hairpin RNA (shRNA) HIV vectors for MITF, and one control shRNA HIV vector (GL3B; for firefly luciferase). An HIV vector expressing two shRNAs for MITF and BRAFV600E was also constructed by tandemly aligning two shRNA expression cassettes in one HIV vector. Seven melanoma cell lines were infected with these shRNA HIV vectors, and the effects of MITF RNAi on the cell growth and apoptosis were evaluated. In six of 7 melanoma cell lines with substantial expression of the MITF protein, the MITF RNAi induced significant inhibition of cell growth, associated with G1 arrest. One melanoma cell line, A375, without any detectable expression of MITF was resistant to the MITF RNAi. One melanoma cell line showed significant apoptotic changes represented by the increase of subG1 population (7.23-7.60%) and increase of cleaved caspase-3 and caspase-9, following the MITF RNAi. Interestingly, the MITF RNAi induced G1 arrest in most melanoma cell lines tested, however, no clear correlation was observed between the protein levels of cdk inhibitors and G1 arrest. Finally, three melanoma cell lines with the simultaneous inhibition of both MITF and BRAFV600E, showed synergistic inhibition of cell growth, which was associated with more G1 arrest.Our results suggest that MITF could be a molecular target for gene therapy for most human melanoma, and the synergistic therapeutic effects of MITF and BRAFV600E indicate the possibly more potent gene therapy for melanoma by simultaneous down-regulation of these molecules.Molecular Therapy (2006) 13, S371–S371; doi: 10.1016/j.ymthe.2006.08.1055 [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) play a key role in innate and adaptive immunity but the access to sufficient amount of DCs for basic and translational research has been limited.We established a novel ex vivo system to develop and expand DCs from hematopoietic stem/progenitor cells (HPCs). Both human and mouse HPCs were expanded first in feeder culture supplemented with c-Kit ligand (KL, stem cell factor, steel factor or CD117 ligand), Flt3 ligand (fms-like tyrosine kinase 3, Flt3L, FL), thrombopoietin (TPO), IL-3, IL-6, and basic fibroblast growth factor (bFGF), and then in a second feeder culture ectopically expressing all above growth factors plus GM-CSF and IL-15.In the dual culture system, CD34+ HPCs differentiated toward DC progenitors (DCPs), which expanded more than five orders of magnitude. The DCPs showed myeloid DC surface phenotype with up-regulation of transcription factors PU.1 and Id2, and DC-related factors homeostatic chemokine ligand 17 (CCL17) and beta-chemokine receptor 6 (CCR6). Multiplex ELISA array and cDNA microarray analyses revealed that the DCPs shared some features of IL-4 and IL-15 DCs but displayed a pronounced proinflammatory phenotype. DCP-derived DCs showed antigen-uptake and immune activation functions analogous to that of the peripheral blood-derived DCs. Furthermore, bone marrow HPC-derived DCP vaccines of tumor-bearing mice suppressed tumor growth in vivo.This novel approach of generating DCP-DCs, which are different from known IL-4 and IL-15 DCs, overcomes both quantitative and qualitative limitations in obtaining functional autologous DCs from a small number of HPCs with great translational potential.

Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAF(V600E). Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAF(V600E) inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAF(V600E) significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment.

Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8+ CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intratumoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.