Dendritic cells are defective in breast cancer patients: a potential role for polyamine in this immunodeficiency

INTRODUCTION: Dendritic cells (DCs) are antigen-presenting cells that are currently employed in cancer clinical trials. However, it is not clear whether their ability to induce tumour-specific immune responses when they are isolated from cancer patients is reduced relative to their ability in vivo. We determined the phenotype and functional activity of DCs from cancer patients and investigated the effect of putrescine, a polyamine molecule that is released in large amounts by cancer cells and has been implicated in metastatic invasion, on DCs. METHODS: The IL-4/GM-CSF (granulocyte–macrophage colony-stimulating factor) procedure for culturing blood monocyte-derived DCs was applied to cells from healthy donors and patients (17 with breast, 7 with colorectal and 10 with renal cell carcinoma). The same peroxide-treated tumour cells (M74 cell line) were used for DC pulsing. We investigated the effects of stimulation of autologous lymphocytes by DCs pulsed with treated tumour cells (DC-Tu), and cytolytic activity of T cells was determined in the same target cells. RESULTS: Certain differences were observed between donors and breast cancer patients. The yield of DCs was dramatically weaker, and expression of MHC class II was lower and the percentage of HLA-DR(-)Lin(- )cells higher in patients. Whatever combination of maturating agents was used, expression of markers of mature DCs was significantly lower in patients. Also, DCs from patients exhibited reduced ability to stimulate cytotoxic T lymphocytes. After DC-Tu stimulation, specific cytolytic activity was enhanced by up to 40% when DCs were from donors but only up to 10% when they were from patients. IFN-γ production was repeatedly found to be enhanced in donors but not in patients. By adding putrescine to DCs from donors, it was possible to enhance the HLA-DR(-)Lin(- )cell percentage and to reduce the final cytolytic activity of lymphocytes after DC-Tu stimulation, mimicking defective DC function. These putrescine-induced deficiencies were reversed by treating DCs with all-trans retinoic acid. CONCLUSION: These data are consistent with blockade of antigen-presenting cells at an early stage of differentiation in patients with breast cancer. Putrescine released in the microenvironmement of DCs could be involved in this blockade. Use of all-trans retinoic acid treatment to reverse this blockade and favour ex vivo expansion of antigen-specific T lymphocytes is of real interest

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the TH1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer

Zoledronic acid renders human M1 and M2 macrophages susceptible to Vδ2(+) γδ T cell cytotoxicity in a perforin-dependent manner.

Vδ2(+) T cells are a subpopulation of γδ T cells in humans that are cytotoxic towards cells which accumulate isopentenyl pyrophosphate. The nitrogen-containing bisphosphonate, zoledronic acid (ZA), can induce tumour cell lines to accumulate isopentenyl pyrophosphate, thus rendering them more susceptible to Vδ2(+) T cell cytotoxicity. However, little is known about whether ZA renders other, non-malignant cell types susceptible. In this study we focussed on macrophages (Mϕs), as these cells have been shown to take up ZA. We differentiated peripheral blood monocytes from healthy donors into Mϕs and then treated them with IFN-γ or IL-4 to generate M1 and M2 Mϕs, respectively. We characterised these Mϕs based on their phenotype and cytokine production and then tested whether ZA rendered them susceptible to Vδ2(+) T cell cytotoxicity. Consistent with the literature, IFN-γ-treated Mϕs expressed higher levels of the M1 markers CD64 and IL-12p70, whereas IL-4-treated Mϕs expressed higher levels of the M2 markers CD206 and chemokine (C-C motif) ligand 18. When treated with ZA, both M1 and M2 Mϕs became susceptible to Vδ2(+) T cell cytotoxicity. Vδ2(+) T cells expressed perforin and degranulated in response to ZA-treated Mϕs as shown by mobilisation of CD107a and CD107b to the cell surface. Furthermore, cytotoxicity towards ZA-treated Mϕs was sensitive-at least in part-to the perforin inhibitor concanamycin A. These findings suggest that ZA can render M1 and M2 Mϕs susceptible to Vδ2(+) T cell cytotoxicity in a perforin-dependent manner, which has important implications regarding the use of ZA in cancer immunotherapy

Aminobisphosphonate-pretreated dendritic cells trigger successful Vgamma9Vdelta2 T cell amplification for immunotherapy in advanced cancer patients.

International audienceHepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vgamma9Vdelta2 T lymphocytes represents an attractive strategy. Indeed, Vgamma9Vdelta2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vgamma9Vdelta2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vgamma9Vdelta2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vgamma9Vdelta2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vgamma9Vdelta2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vgamma9Vdelta2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vgamma9Vdelta2 T cells as measured by IFN-gamma production. Moreover, we demonstrated that the cytotoxic level of Vgamma9Vdelta2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vgamma9Vdelta2 T cells leads eligible for Vgamma9Vdelta2 T cell adoptive immunotherapy the HCC and mCRC patients

The MHC class Ib protein ULBP1 is a nonredundant determinant of leukemia/lymphoma susceptibility to γδ T-cell cytotoxicity

© 2010 by The American Society of HematologyOn the path to successful immunotherapy of hematopoietic tumors, γδ T cells offer great promise because of their human leukocyte antigen (HLA)–unrestricted targeting of a wide variety of leukemias/lymphomas. However, the molecular mechanisms underlying lymphoma recognition by γδ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to γδ T cell–mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all Vγ9+ T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to γδ T cell–based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials.This work was distinguished with the Pfizer Award for ClinicalResearch 2009. This work was further supported by Fundação para a Ciência e Tecnologia (PTDC/SAU-MII/71662/2006) and Fundação Calouste Gulbenkian (SDH Oncologia 2008; Projecto 99293). L.F.M. is a Young Investigator from the Human Frontier Science Program and was supported by Fundação Luso-Americana para o Desenvolvimento and Fundação para a Ciência e Tecnologia (FCT; PTDC/SAU-MII/69280/2006 and PTDC/SAU-MII/78333/2006). T.L. and D.V.C. received PhD fellowships (47342/2008 and 37898/2007) from FCT

Clinical evaluation of autologous gamma delta T cell-based immunotherapy for metastatic solid tumours

BACKGROUND: Adoptive transfer of ex vivo expanded autologous V gamma 9V delta 2 T cells may be of therapeutic benefit for cancer because of their potent direct cytotoxicity towards tumour cells, synergistic cytotoxicity when combined with aminobisphosphonates and enhancement of antibody-dependent cell-mediated cytotoxicity