Nourredine Himoudi

Human γδ T Lymphocytes Are Licensed for Professional Antigen Presentation by Interaction with Opsonized Target Cells

Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αβ T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner.

Clusterin, a haploinsufficient tumor suppressor gene in neuroblastomas.

BACKGROUND Clusterin expression in various types of human cancers may be higher or lower than in normal tissue, and clusterin may promote or inhibit apoptosis, cell motility, and inflammation. We investigated the role of clusterin in tumor development in mouse models of neuroblastoma. METHODS We assessed expression of microRNAs in the miR-17-92 cluster by real-time reverse transcription-polymerase chain reaction in MYCN-transfected SH-SY5Y and SH-EP cells and inhibited expression by transfection with microRNA antisense oligonucleotides. Tumor development was studied in mice (n = 66) that were heterozygous or homozygous for the MYCN transgene and/or for the clusterin gene; these mice were from a cross between MYCN-transgenic mice, which develop neuroblastoma, and clusterin-knockout mice. Tumor growth and metastasis were studied in immunodeficient mice that were injected with human neuroblastoma cells that had enhanced (by clusterin transfection, four mice per group) or reduced (by clusterin short hairpin RNA [shRNA] transfection, eight mice per group) clusterin expression. All statistical tests were two-sided. RESULTS Clusterin expression increased when expression of MYCN-induced miR-17-92 microRNA cluster in SH-SY5Y neuroblastoma cells was inhibited by transfection with antisense oligonucleotides compared with scrambled oligonucleotides. Statistically significantly more neuroblastoma-bearing MYCN-transgenic mice were found in groups with zero or one clusterin allele than in those with two clusterin alleles (eg, 12 tumor-bearing mice in the zero-allele group vs three in the two-allele group, n = 22 mice per group; relative risk for neuroblastoma development = 4.85, 95% confidence interval [CI] = 1.69 to 14.00; P = .005). Five weeks after injection, fewer clusterin-overexpressing LA-N-5 human neuroblastoma cells than control cells were found in mouse liver or bone marrow, but statistically significantly more clusterin shRNA-transfected HTLA230 cells (3.27%, with decreased clusterin expression) than control-transfected cells (1.53%) were found in the bone marrow (difference = 1.74%, 95% CI = 0.24% to 3.24%, P = .026). CONCLUSIONS We report, to our knowledge, the first genetic evidence that clusterin is a tumor and metastasis suppressor gene.

Coordinated oncogenic transformation and inhibition of host immune responses by the PAX3-FKHR fusion oncoprotein

Tumors have evolved elaborate mechanisms for evading immune detection, such as production of immunoinhibitory cytokines and down-regulation of major histocompatibility complex (MHC) expression. We have studied PAX3-FKHR as an example of an oncogenic fusion protein associated with an aggressive metastatic cancer. We show that PAX3-FKHR alters expression of genes that are normally regulated by Janus kinase/signal transducer and activator of transcription (STAT) signaling pathways. This occurs as a result of a specific interaction between PAX3-FKHR and the STAT3 transcription factor, which results in a dramatic reduction in tumor MHC expression, and an alteration in local cytokine concentrations to inhibit surrounding inflammatory cells and immune detection. Collectively, these data show that an oncogenic transcription factor can promote tumor growth and tissue invasion while inhibiting local inflammatory and immune responses. This is the first time that an immunomodulatory role has been described for an oncogenic fusion protein.

Lack of T-cell responses following autologous tumour lysate pulsed dendritic cell vaccination, in patients with relapsed osteosarcoma.

BackgroundImmunotherapy using autologous dendritic cell (DC) vaccination has not been systematically evaluated in osteosarcoma. We therefore conducted a phase I trial to assess feasibility, safety and tumour-specific immune responses in patients with relapsed disease.Patients and methodsOf 13 recruited patients with relapsed osteosarcoma, 12 received 3 weekly vaccines of autologous DCs matured with autologous tumour lysate and keyhole limpet haemocyanin (KLH), to a maximum of 6 vaccinations. An additional 3 paediatric patients afflicted with other tumour types and with relapsed disease received vaccines generated with identical methodology. Immune responses were assessed using an ELISpot assay for the detection of interferon gamma, whilst interleukin-2 and granzyme B were additionally assessed in cases where interferon-γ responses were induced.ResultsIn total 61 vaccines, of homogeneous maturation phenotype and viability, were administered with no significant toxicity. Only in 2 out of 12 treated osteosarcoma cases was there an induction of specific T-cell immune response to the tumour, whilst a strong but non-specific immune response was induced in 1 further osteosarcoma patient. Immune response against KLH was induced in only 3 out of 12 osteosarcoma patients. In contrast, three additional non-osteosarcoma patients showed significant T-cell responses to vaccine.ConclusionWe have shown the strategy of DC vaccination in relapsed osteosarcoma is safe and feasible. However, significant anti-tumour responses were induced in only 2 out of 12 vaccinated patients with no evidence of clinical benefit. Comparison of results with identically treated control patients suggests that osteosarcoma patients might be relatively insensitive to DC-based vaccine treatments.

Increased PRAME antigen-specific killing of malignant cell lines by low avidity CTL clones, following treatment with 5-Aza-2′-Deoxycytidine

The cancer testis antigen Preferentially Expressed Antigen of Melanoma (PRAME) is overexpressed in many solid tumours and haematological malignancies whilst showing minimal expression in normal tissues and is therefore a promising target for immunotherapy. HLA-A0201-restricted peptide epitopes from PRAME have previously been identified as potential immunogens to drive antigen-specific autologous CTL responses, capable of lysing PRAME expressing tumour cells. CTL lines, from 13 normal donors and 10 melanoma patients, all of whom were HLA-A0201 positive, were generated against the PRAME peptide epitope PRA100−108. Specific killing activity against PRA100−108 peptide-pulsed targets was weak compared with CTL lines directed against known immunodominant peptides. Moreover, limiting dilution cloning from selected PRAME-specific CTL lines resulted in the generation of a clone of only low to intermediate avidity. Addition of the demethylating agent 5-aza-2′-Deoxycytidine (DAC) increased PRAME expression in 7 out of 11 malignant cell lines including several B lineage leukaemia lines and also increased class I expression. Pre-treatment of target cells was associated with increased sensitivity to antigen-specific killing by the low avidity CTL. When CTL, as well as of the target cells, were treated, the antigen-specific killing was further augmented. Interestingly, one HLA-A0201-negative DAC-treated line (RAJI) showed increased sensitivity to killing by clones despite a failure of expression of PRAME or HLA-A0201. Together these data point to a general increased augmentation of cancer immunogenocity by DAC involving both antigen-specific and non-specific mechanisms.

Bone Marrow-Derived IFN-Producing Killer Dendritic Cells Account for the Tumoricidal Activity of Unpulsed Dendritic Cells

The CD11cintB220+NK1.1+CD49+ subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11cintB220+NK1.1+CD49+ cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 × 103 highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.

Development of Cellular Immune Responses against PAX5, a Novel Target for Cancer Immunotherapy

PAX5 is a member of the PAX family of developmental transcription factors with an important role in B-cell development. Its expression in normal adult tissue is limited to the hemopoietic system, but it is aberrantly expressed in a number of solid cancers and leukemias where it functions as an oncogene. We therefore hypothesized that anti-PAX5 immune responses could be used to target a number of malignancies without significant toxicity. We screened PAX5 peptides for the ability to bind HLA-A2 and identified a novel sequence, TLPGYPPHV (referred to as TLP). CTL lines against TLP were generated from peripheral blood of five normal HLA-A2-positive blood donors and showed specific HLA-A2-restricted killing against PAX5-expressing target cells. We generated high-avidity CTL clones from these lines capable of killing cells pulsed with <1 nmol/L of TLP and killing a range of PAX5-expressing malignant cell lines. I.v. injection of an anti-PAX5 CTL clone into immunodeficient mice bearing s.c. human tumors resulted in specific growth inhibition of PAX5-expressing tumors. This knowledge can be used for the therapeutic generation of CTL lines or the cloning of high-avidity T-cell receptor genes for use in adoptive immunotherapy.

Migratory and Antigen Presentation Functions of IFN-Producing Killer Dendritic Cells

The CD11c(int) B220(+) NK1.1(+) CD49(+) subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties with both dendritic cells and natural killer cells. We have previously shown that IKDCs within murine bone marrow-derived DC preparations are essential for the antitumor activity of unpulsed DCs. Here we show that bone marrow-derived IKDCs (BM-IKDC) migrate in vivo into tumors and thence to tumor draining lymph nodes, where they highly express MHC class II and costimulatory molecules. In vitro, freshly isolated BM-IKDCs, fluorescence-activated cell sorted to homogeneity, have no intrinsic antigen presentation function unless cocultured with tumor target cells. On killing of target cells, they can cross-present antigens to stimulate antigen-primed CD8 T cells and can also present antigens to antigen-primed CD4 cells. In vivo, in mice lacking class I-restricted antigen-presenting cell function, robust proliferation of antigen-specific T cells is achieved after adoptive transfer of BM-IKDCs at an injection site distant to the tumor site. Therefore, BM-IKDCs are capable of cytotoxic killing of tumor targets and also of potent antigen presentation after encountering antigen in the context of a viable target cell.

Development of anti-PAX3 immune responses; a target for cancer immunotherapy

PAX3 is overexpressed in several human cancers and is absent from normal adult human tissues. It is known to have an oncogenic function in human malignancy, and is therefore a promising target for cancer immunotherapy. We screened the murine and human PAX3 amino acid sequences for peptides that bind common MHC class I types, and identified murine GVFINGRPL and human KLTEARVQV sequences. Mice immunised with either a selected PAX3 peptide, or with a PAX3 expressing DNA vector, developed specific anti-PAX3 immune responses that inhibited tumour growth. The intensity of the immune response was significantly enhanced by pulsing of the peptide onto dendritic cells. Anti-PAX3 T cell lines were established from splenocytes of immunised mice. Intravenous administration of anti-PAX3 T cells caused regression of established tumours indicating a promising clinical application for anti-PAX3 immunotherapy. The human peptide stimulated growth of similar T cell lines from peripheral blood of three out of three normal human blood donors. These showed specific cytotoxicity against a range of human PAX3+ and HLA-A2+ cancer cell lines. Moreover, an anti-PAX3 response was detected as a component of the anti-tumour immune response in a patient treated with lysate pulsed dendritic cell vaccination. The ability to generate strong and specific anti PAX3 immune responses from the T cell repertoire in both mice and humans, provides evidence for PAX3 as a promising target for immunotherapy of cancer.

An Optimized GD2-Targeting Retroviral Cassette for More Potent and Safer Cellular Therapy of Neuroblastoma and Other Cancers

Neuroblastoma is the commonest extra cranial solid cancer of childhood. Despite escalation of treatment regimens, a significant minority of patients die of their disease. Disialoganglioside (GD2) is consistently expressed at high-levels in neuroblastoma tumors, which have been targeted with some success using therapeutic monoclonal antibodies. GD2 is also expressed in a range of other cancer but with the exception of some peripheral nerves is largely absent from non-transformed tissues. Chimeric Antigen Receptors (CARs) are artificial type I proteins which graft the specificity of a monoclonal antibody onto a T-cell. Clinical data with early CAR designs directed against GD2 have shown some promise in Neuroblastoma. Here, we describe a GD2-targeting CAR retroviral cassette, which has been optimized for CAR T-cell persistence, efficacy and safety.