Jean-Paul Molens

Virus or TLR Agonists Induce TRAIL-Mediated Cytotoxic Activity of Plasmacytoid Dendritic Cells

Among dendritic cells, plasmacytoid dendritic cells (PDC) represent a functionally distinct lineage. Regarding innate immunity, PDC secrete large amounts of type I IFN upon viral exposure or stimulation by microbial products such as unmethylated CpG-motif containing oligo-DNA due to their selective expression of TLR7 and TLR9. We asked whether they could acquire cytotoxic functions during the early phases of infection or after activation with TLR7 or TLR9 agonists. In the present study, we describe a human PDC cell line called GEN2.2, derived from leukemic PDC, that shares most of the phenotypic and functional features of normal PDC. We show that after contact with the influenza virus, GEN2.2, as well as normal PDC, acquires TRAIL and killer activity against TRAIL-sensitive target cells. Moreover, we show that activation of GEN2.2 cells by CpG-motif containing oligo-DNA or R848 also induces TRAIL and endows them with the ability to kill melanoma cells. Therefore, PDC may represent a major component of innate immunity that could participate to the clearance of infected cells and tumor cells. This phenomenon could be relevant for the efficacy of TLR7 or TLR9 agonists in the therapy of infectious disease and cancer.

Functional expression of CD80 and CD86 allows immunogenicity of malignant B cells from non-Hodgkin’s lymphomas

We analyzed the accessory function of malignant B cells from non-Hodgkins lymphomas (NHLs). Among the 70 samples of malignant B cells included, four patterns of expression of the costimulatory molecules CD80 and CD86 were distinguished (+/+, +/-, -/+ and -/-). In two-thirds of the cases, CD80, CD86, or both were expressed. To investigate the relevance of these molecules for tumor immunogenicity, mixed lymphocyte reactions (MLR) were performed with allogeneic responding T cells and malignant B cells from nine NHL patients. Regardless of the level of expression of CD80 and CD86, significant proliferation was induced in the responder cells. The addition of monoclonal antibodies directed against CD80 and CD86 at the beginning of MLR almost completely inhibited this proliferation. We show that, during MLR, a high level of expression of CD80 and CD86 was induced in NHL B cells. Thus, cooperation between responding and stimulator cells seems to occur during MLR, allowing induction of optimal accessory function of B cells. We investigated whether malignant B cells cultured with CD40-L-transfected L cells in the presence of IL-4 could augment their antigen-presenting cell (APC) functions. The culture of NHL B cells in this sytem induced strong upregulation of the expression of CD80 and CD86 as well as other molecules involved in accessory cell functions (HLA class I, CD54, and CD58). In half of the cases, this activation resulted in enhanced proliferation of allo-T cells as compared to the proliferation induced by nonactivated malignant B cells. Our results show that NHL B cells are able to express functional CD80 and CD86 and to be fully competent APC. This suggests that the absence of an efficient T cell-mediated antitumor response in vivo is not related to a deficiency in the APC functions of malignant B cells.

Differentiation of antitumor-specific cytotoxic T lymphocytes from autologous tumor infiltrating lymphocytes in non-Hodgkin’s lymphomas

The present study describes a new culture protocol allowing the activation and proliferation of autologous tumor infiltrating T lymphocytes (TIL), and the generation of antitumor specific CTL in non-Hodgkins lymphoma (NHL). Cells from eight patients with indolent NHL were used. We performed 3-week co-cultures of TIL with irradiated autologous malignant B cells in the presence of low doses of IL-1beta, IL-2 and IL-12. The proliferation, phenotype and cytotoxicity, and antitumor specificity of T cells recovered were studied. T-cell clonality was analyzed using TCRgamma gene rearrangement amplification by a multiplex PCR. Under these culture conditions, TIL proliferated, and the CD8+ T lymphocytes that were in a minority at the beginning of the culture increased dramatically in 6 out of 8 cases. In two cases, CD4+ T lymphocytes expanded. We showed that an oligoclonal selection of reactive T cells occurred in culture. Specific cytotoxicity developed against autologous malignant B cells in the 6 cases where there was an expansion of CD8+ T lymphocytes. Inhibition experiments performed with mAb directed against HLA class I and II molecules, CD4, CD8 and TCRgammadelta showed that the cytotoxic effector cells were CD8+ T lymphocytes probably expressing TCRalphabeta+. Cytokine secretion was analyzed in culture medium, and we detected significant levels of IFN-gamma, TNF-alpha, and IL-10 and no IL-4 (except in one case). Our results demonstrate that memory T cells from lymphoma patients can be amplified and differentiated into antitumor cytotoxic cells using a combination of the cytokines IL-1beta, IL-2, and IL-12 in association with non modified tumor cells.

Photochemotherapy Induces the Apoptosis of Monocytes Without Impairing Their Function

Background. Extracorporeal photopheresis (ECP) is a powerful therapy currently used to treat various hematological disorders as in graft versus host disease. Clinical data clearly demonstrate its efficacy and immunomodulation toward the pathogenic T cells. However, ECP mechanism of action is still poorly understood. Monocytes represent up to 30% of the total amount of treated cells and are known to play an important role in adaptive immunity. However, data from previous reports analyzing the effect of psoralen and UV-A irradiation (PUVA) on their functions are heterogeneous. In this study, we focused on the effect of PUVA on human monocytes functions in adaptive immunity. Design and Methods. Purified human monocytes were treated in vitro by PUVA. We measured their kinetic of apoptosis after the treatment. We also determine whether their phenotype and functionalities were modified. Finally, we assessed the functionalities of PUVA-treated monocytes-derived dendritic cells (DC). Results. PUVA treatment sentenced purified monocytes to die in 6 days and immediately altered their migratory capacities without impairing their ability of endocytosis. It also up-regulated co-stimulatory molecules and production of inflammatory cytokines on activation and consequently stimulated allogeneic or autologous T cells as efficiently as untreated monocytes. Moreover, PUVA-treated monocytes retained their ability to differentiate into fully functional DC that maturated and stimulated T cells as well as normal DC. Conclusions. Our data demonstrate that monocytes undergo apoptosis and loose a part of their migratory capacity after ECP and the surviving cell functionalities are not impaired, suggesting that monocytes have a minor effect on ECP-mediated immunomodulation.

Impairment of death-inducing signalling complex formation in CD95-resistant human primary lymphoma B cells

Multiple mechanisms exist by which tumour cells can escape CD95‐mediated apoptosis. Previous studies by our laboratory have shown that primary B cells from non‐Hodgkins Lymphoma (B‐NHL) were resistant to CD95‐induced cell death. In the current study, we have analysed the mechanisms underlying CD95 resistance in primary human lymphoma B cells. We report that FADD (FAS‐associated death domain protein) and caspase‐8 were constitutively expressed in lymphoma B cells and that the CD95 pathway was blocked upstream to caspase‐8 activation. However, caspase‐8 was processed and functional after treatment with staurosporine (STS). We found that the expression levels of FLICE (FADD‐like interleukin‐1 beta‐converting enzyme)‐Inhibitory Protein (c‐FLIP) and Bcl‐2‐related proteins were heterogeneous in B‐NHL cells and were not related to CD95 resistance. Finally, we report the absence of a CD95‐induced signalling complex [death‐inducing signalling complex (DISC)] in lymphoma B cells, with no FADD and caspase‐8 recruitment to CD95 receptor. In contrast, DISC formation was observed in CD95‐resistant non‐tumoural (NT) B cells. Therefore, we propose that the absence of DISC formation in primary lymphoma B cells may contribute to protect these cells from CD95‐induced apoptosis.

Differentiation of anti-tumour cytotoxic T lymphocytes from autologous peripheral blood lymphocytes in non-Hodgkin's lymphomas

Summary. We have previously reported that specific anti‐tumour cytotoxic T cells (CTL) can be differentiated from tumour‐infiltrating lymphocytes (TIL) in non‐Hodgkins lymphoma. We found that the combination of interleukin (IL)‐1, IL‐2 and IL‐12 was very efficient for expansion of CD8+ T‐cell receptor (TCR)αβ+ T cells and for development of their ability to specifically lyse tumour cells. In this study, we investigated whether anti‐tumour T cells could be generated from the peripheral blood of patients using the culture protocol developed for TIL. Autologous T cells and tumour B cells from five patients were included in this study. It was found that polyclonal anti‐tumour cytotoxic effector cells were generated when cultured in the presence of IL‐1β, IL‐2 and IL‐12. Interestingly, tumour cells were lysed by perforin/granzyme‐mediated cytolysis and not by CD95‐mediated apoptosis. By performing inhibition experiments, it was observed that both CD8+ and CD4+ T cells were responsible for the cytotoxic effect and that they were able to recognize malignant B cells by either a major histocompatibility complex (MHC)‐restricted or MHC‐non‐restricted mechanism. Intriguingly, in addition to interferon‐γ and tumour necrosis factor‐α, IL‐10 was secreted continuously during culture. The source of patient T cells used for the generation of anti‐tumour CTL should be based on the results obtained with peripheral blood lymphocytes and TIL.

Relationships Between Susceptibility to LAK Cell-Mediated Lysis, Conjugate Formation and Expression of Adhesion Molecules in B-Cell Derived Non-Hodgkin's Lymphomas

Adoptive immunotherapy with LAK cells has been investigated for the treatment of B-cell-derived lymphomas, but only a few significant tumor regressions were obtained. In order to explain this refractory state, the sensitivity to normal LAK-mediated lysis of 30 non-Hodgkins lymphoma (NHL) malignant B-cells was determined using flow cytofluorimetry. A large heterogeneity was found, and we report a close correlation (p < 0.001) between the extent of lysis of malignant B-cells and their ability to form conjugates with LAK cells; which is the first step in LAK-mediated cytolysis. The levels of expression of HLA class I molecules, LFA-1 (CD11a/CD18), CD54 and CD58 were also studied and found to be expressed very heterogeneously. CD54 expression on malignant B-cells plays a major role in the initial conjugate formation with LAK cells (p < 0.001), and this was confirmed by inhibition experiments. Our results suggest that a weak expression of CD54 could constitute one mechanism by which NHL tumor B-cells escape natural immune surveillance and resist LAK cells immunotherapies.

From the Study of Tumor Cell Immunogenicity to the Generation of Antitumor Cytotoxic Cells in Non-Hodgkin's Lymphomas

The question of the immunogenicity of non-Hodgkins lymphoma (NHL) B cells has been investigated in an attempt to support the development of new immunotherapeutic treatments for this disorder, which remains resistant to conventional treatments in most cases. In the present review, we report and discuss our new findings in the field of NHL B cell immunogenicity. One aspect of our work is the description of the expression and functions of membrane molecules associated with antigen presentation. The expression levels of adhesion molecules was measured, and the relevance of this expression to the sensitivity of malignant B cells to cell-mediated lysis was studied. Since the T cell response relies on the expression of both HLA class I and II molecules, we also investigated whether or not these molecules were present at the surface of NHL B cells. Subsequently, we asked whether antitumor CTL and LAK cells could be developed and analyzed the mechanisms of cell lysis involved. Since the generation of a T cell response requires the expression of the costimulatory molecules CD80 and CD86, we investigated their in vivo expression and their modulation in vitro during contact with responding T lymphocytes. The understanding of the immunogenicity of NHL B cells has enabled us to develop a new culture protocol to induce antitumor specific autologous CTL. The originality of NHL B cells -unlike most other tumor cells- is to be able to function as antigen presenting cells (APC) and to activate a T cell response in the absence of other professional APC. Over the next few years, these findings should allow the generation of anti-NHL specific T cells for adoptive immunotherapy and for the identification of NHL-associated antigens.

Abstract 3686: Generation of an off-the-shelf cell-based platform (PDC-vac) for active antitumor immunotherapy

The development of efficient vaccines for the treatment of cancers in combination or not with other agents such as checkpoint inhibitors represents a major public health issue but remains a challenge. A dozen of vaccine drug products is expected to be marketed in the next few years. In this context, off-the-shelf and potent platforms are more attractive than individualized vaccines that are tailored to each patient in many aspects. We have recently developed a new cell-based vaccine approach named PDC*vac (formerly GeniusVac). This cell-based vaccine was developed thanks to the establishment, from leukemic PDC, of unique human PDC line (PDC*line). We have previously shown the potential of peptide-loaded PDC*line to induce strong and fully functional HLA-A*02:01-restricted anti-tumor immunity in vitro and in vivo (Aspord et al, 2010, 2012). PDC*mel (GenusVac-Mel4), our first product is currently developed for the treatment of melanoma patients (NCT01863108). The high efficiency of PDC*vac renders the strategy very attractive for further clinical developments in active immunotherapy field. Our aim is to optimize PDC*vac technology by passing use peptides and render the vaccine easier to manufacture. We engineered our PDC*line using retrovirus to force the endogenous expression of one or more tumor or viral antigenic peptides or whole tumor/viral proteins (gp100, tyrosinase, Melan-A, Mage-A3, CMVpp65, flu, EBV) that broaden the panel of antigens presented. Data obtaining with different polypeptide and protein gene coding constructions and the expansion of multi-specific antigen-specific T-cells at the same time will be presented. Moreover, we will present how we succeed to obtain 100% of efficacy in antigen presentation by the PDC*line engineered to express tumor proteins or polyepitopes. We have validated the use of retrovirus constructs to create a new generation of an “off-the-shelf” cancer vaccines highly potent to stimulate antitumor-specific T-cells. Citation Format: Joel Plumas, Kevin Lenogue, Alexandre Walencik, Jean-Paul Molens, Laurence Chaperot, Martin Pule. Generation of an off-the-shelf cell-based platform (PDC-vac) for active antitumor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2017-3686