Induction of antiviral cytotoxic T cells by plasmacytoid dendritic cells for adoptive immunotherapy of posttransplant diseases.

Virus-associated hematologic malignancies (EBV lymphoproliferative disease) and opportunistic infections (CMV) represent a major cause of hematopoietic stem cell and solid organ transplantation failure. Adoptive transfer of antigen-specific T lymphocytes appears to be a major and successful immunotherapeutic strategy, but improvements are needed to reliably produce high numbers of virus-specific T cells with appropriate requirements for adoptive immunotherapy that would allow extensive clinical use. Since plasmacytoid dendritic cells (pDCs) are crucial in launching antiviral responses, we investigated their capacity to elicit functional antiviral T-cell responses for adoptive cellular immunotherapy using a unique pDC line and antigens derived from Influenza, CMV and EBV viruses. Stimulation of peripheral blood mononuclear cells from HLA-A*0201(+) donors by HLA-A0201 matched pDCs pulsed with viral-derived peptides triggered high levels of multi-specific and functional cytotoxic T-cell responses (up to 99% tetramer(+) CD8 T cells) in vitro. Furthermore, the central/effector memory cytotoxic T cells elicited by the pDCs strongly display antiviral activity upon adoptive transfer into a humanized mouse model that mimics a virus-induced malignancy. We provide a simple and potent method to generate virus-specific CTL with the required properties for adoptive cellular immunotherapy of post-transplant diseases.

[GENiusVac, a novel antitumor vaccine strategy based on allogeneic plasmacytoid dendritic cells]. / GENiusVac, une stratégie vaccinale antitumorale innovante basée sur des cellules dendritiques plasmocytoïdes allogéniques.

The development of effective vaccines against cancer and viruses still remains a challenge. Many immunotherapeutic strategies have been developed but without sufficient therapeutic success. Plasmacytoid dendritic cells (pDC) play a crucial role in antitumor and antiviral responses. Despite their outstanding functional properties, their therapeutic potential has not yet been worked out. We propose a new immunotherapeutic strategy based on a pDC cell line irradiated and pulsed with tumor or viral antigens. GENiusVac allows the induction of multispecific and highly functional cytotoxic cell responses directed against viral or tumor targets. We demonstrated the potential of this strategy in vitro, its therapeutic efficacy in vivo in a humanized mouse model, and its clinical relevance ex vivo from melanoma patients' cells. GENiusVac highlights pDCs as potent vector of immunotherapy and provide a way to exploit them in cell therapy to fight cancer or chronic viral infections.

[Extracorporeal photochemotherapy or immunotherapy using cells modified by photochemistry]. / La photochimiothérapie extracorporelle ou l'immunothérapie par cellules modifiées par photochimie.

Extracorporeal photochemotherapy (ECP) is an autologous cell therapy used for the treatment of diseases involving pathogenic cells: cutaneous T-cell lymphoma, organ rejection and graft versus host disease. During an ECP procedure, patients receive a cellular product consisting of autologous mononuclear cells, containing the pathogenic cells, treated with a photosensitising agent and an UV-A radiation. The aim of the treatment is to induce a specific immune reaction modulating the activity of untreated pathogenic lymphocytes responsible for the disease and therefore an improvement of clinical manifestations. The precise mechanisms of action remain to be defined in humans. Its efficacy coupled with the absence of side effects could lead to decrease the use of immunosuppressive drugs. PCE appears as an immunotherapy using cells modified by photochemistry, which allows specific immune modulation of pathogenic lymphocytes.

Influenza recombinant vaccine: matrix protein M1 on the platform of the adenovirus dodecahedron.

We propose a novel influenza vaccine composed of the adenovirus dodecahedron (Dd) as delivery platform carrying an internal influenza matrix protein M1. To attach the antigen to the vector we used WW domains interacting with Dd. Successful internalization of the Dd-M1WW complex was observed using biochemical and cell biology techniques. We show here that the complex of Dd with antigen is a potent activator of human myeloid dendritic cells (MDC), and that it is efficiently presented by MDC to M1-specific CD8+ T lymphocytes. These results show that proposed vaccine model is feasible and that adenovirus dodecahedron is a potent delivery platform for foreign antigens to human cells.

Efficient characterization of the TCR repertoire in lymph nodes by flow cytometry.

Analysis of the T-cell receptor (TCR) repertoire by flow cytometry proved to be relevant for investigating T-cell diversity and detecting reactive cells in blood samples. We used this approach to characterize non-malignant T-lymphocytes in lymph nodes and give insights into their origin. The TCR repertoire of CD4+ and CD8+ T-cells from 81 lymph nodes was analyzed with a four-color flow cytometer using a wide panel of 25 anti-Vbeta monoclonal antibodies. Flow cytometry proved to be a useful and informative technique. We demonstrated a diversified TCR-Vbeta repertoire, and only low level expansions, in 53% of the samples. They involved nearly all Vbeta families, were more frequent in the CD8+ subset of older patients, but were not related to pathology. No evidence could be demonstrated in favor of stimulation by common antigens. Interestingly, the TCR-Vbeta repertoire proved to be very similar in lymph nodes and blood samples. Our results argue that in the cases studied, lymph node enlargement is mainly due to an increased homing of circulating T-cells. They also provide reference values for expression of 25 TCR-Vbeta in lymph nodes, which could serve as a basis for further applications in diagnosis of T-cell lymphoproliferative disorders.

Recurrent genomic aberrations combined with deletions of various tumour suppressor genes may deregulate the G1/S transition in CD4+CD56+ haematodermic neoplasms and contribute to the aggressiveness of the disease.

CD4+CD56+ haematodermic neoplasms (HDN) constitute a rare disease characterized by aggressive clinical behaviour and a poor prognosis. Tumour cells from HDN are leukaemic counterparts of plasmacytoid dendritic cells (pDCs). Despite increased knowledge of the ontogenetic origin of these tumours, the genetic causes and oncogenic signalling events involved in malignant transformation are still unknown. To delineate novel candidate regions and disease-related genes, we studied nine typical CD4+CD56+ HDN cases using genome-wide high-resolution array comparative genomic hybridization (CGH). Genomic imbalances, which were predominantly losses, were frequently detected. Gross genomic losses or gains involving an entire chromosome were observed in eight cases. The most frequent imbalances were deletions of chromosome 9, chromosome 13 and partial losses affecting 17p or 12p. Combinations of deletions of tumour suppressor genes (TSG), namely RB1, CDKN1B (p27), CDKN2A, (p16(ink4a), p14(arf)) or TP53 (p53), were observed in all cases. These results indicate that deletion events altering G1/S regulation are crucial for HDN oncogenesis. Furthermore, in addition to frequent sporadic gene losses, in one case we observed a 8q24 interstitial deletion that brought MYC closer to miR-30b/miR-30d, which may be related to their deregulation. Taken together, these results indicate that in addition to frequent G1/S checkpoint alterations, various genetic events could contribute to the chemoresistance of the tumour.

Whole lymphoma B cells allow efficient cross-presentation of antigens by dendritic cells.

BACKGROUND: In order to compensate for the paucity of defined tumor antigens (Ag) in non-Hodgkin's lymphomas, a promising approach might be the use of whole tumor cells as a source of tumor Ag to pulse antigen-presenting cells (APC). However, it is not presently known how the tumor cells should be delivered to APC to optimize the cross-presentation of tumor Ag to anti-tumor CD8 T cells. We aimed to compare CD20-opsonized, apoptotic and necrotic human tumor cells for their capacity to induce endocytosis and cross-presentation of tumor-associated Ag by dendritic cells (DC) or macrophages. METHODS: Endocytosis of human tumor-derived material by macrophages or DC was monitored by flow cytometry. We used a previously described influenza model and studied cross-presentation of viral Ag as cellular surrogate tumor-associated Ag by APC after endocytosis of lymphoma B cells treated by inactivated influenza virus. RESULTS: Optimal endocytosis was obtained when tumor cells were opsonized by an anti-CD20 antibody and, as expected, macrophages were more phagocytic than DC. However, Ag from opsonized, apoptotic and live cells, but not from necrotic lymphoma cells, were efficiently cross-presented by DC but not by macrophages. DISCUSSION: We have developed a new model with human primary lymphoma cells to study the cross-presentation of tumor-associated Ag by APC. The results we have obtained support the use of whole lymphoma cells from patients to pulse DC to induce an anti-tumor immune response.

E2F1 induces apoptosis and sensitizes human lung adenocarcinoma cells to death-receptor-mediated apoptosis through specific downregulation of c-FLIP(short).

E2F1 is a transcription factor that plays a well-documented role during S phase progression and apoptosis. We had previously postulated that the low level of E2F1 in primary lung adenocarcinoma contributes to their carcinogenesis. Here, we show that E2F1 triggers apoptosis in various lung adenocarcinoma cell lines by a mechanism involving the specific downregulation of the cellular FLICE-inhibitory protein short, leading to caspase-8 activation at the death-inducing signaling complex. Importantly, we also provide evidence that E2F1 sensitizes tumor as well as primary cells to apoptosis mediated by FAS ligand or tumor necrosis factor-related apoptosis-inducing ligand, and enhances the cytotoxic effect of T lymphocytes against tumor cells. Finally, we describe the specific overexpression of c-FLIP(S) in human lung adenocarcinomas with low level of E2F1. Overall, our data identify E2F1 as a critical determinant of the cellular response to death-receptor-mediated apoptosis, and suggest that its downregulation contributes to the immune escape of lung adenocarcinoma tumor cells.

Mesenchymal stem cells induce apoptosis of activated T cells.

Mesenchymal stem cells (MSC) have recently been used successfully in humans to control severe graft-versus-host disease. However, the mechanisms involved in their immunomodulatory effects remain a matter of debate. Here, we show that MSC are unable to activate allogeneic T cells even in the presence of T-cell growth factors. We then found that MSC inhibit T-cell proliferation triggered either by allogeneic, mitogenic or antigen-specific stimuli. Interestingly, MSC inhibit T-cell proliferation by inducing apoptosis of activated T cells, but have no effect on resting T cells. Furthermore, we show that this apoptosis could be related to the conversion of tryptophan into kynurenine by indoleamine 2,3-dioxygenase expressed by MSC in the presence of IFNgamma. Moreover, we show that the inhibitory effect of MSC is neither abrogated nor modified during expansion in culture or after irradiation. Together, these results bring new insight to the mechanisms of immunosuppression induced by MSC and might help to develop their clinical use controlling immune-related adverse effects in humans.

In situ leukemic plasmacytoid dendritic cells pattern of chemokine receptors expression and in vitro migratory response.

Plasmacytoid dendritic cell (PDC) leukemia/lymphoma is a rare neoplasm presenting cutaneous lesions at the time of diagnosis, followed by dissemination to bone marrow, lymph nodes, and other lymphoid and nonlymphoid organs. Since these leukemic counterparts of human PDC are similar to normal PDC, we studied their chemokine receptor equipment and their migratory capacities. We found both in skin lesions and in invaded lymph nodes an expression by tumor cells of CXCR3, CXCR4, and CCR7, and the concomitant expression by cells in the microenvironment of their respective ligands CXCL9, CXCL12, and CCL19. Moreover, flow cytometry phenotype of leukemic PDC (LPDC) revealed an unexpected expression of CCR6. We show that fresh tumor cells are able to migrate in response to CXCR4, CCR2, CCR5, CCR6, and CCR7 ligands, and the ability of CXCR3 ligands to increase the responsiveness to CXCL12. IL-3- or virus-induced activation of LPDC leads to downregulation of CXCR3 and CXCR4, and upregulation of CCR7, associated with the loss of response to CXCL12, and the acquisition of sensitivity to CCL19. Altogether, these results suggest that the preferential accumulation of LPDC in the skin or lymph nodes could be orchestrated by CXCR3, CXCR4, CCR6, and CCR7 ligands, found in nontumoral structures of invaded organs.

Preparation of purified lymphoma cells suitable for therapy.

BACKGROUND: Very few tumoral Ags have yet been isolated in NHL B cells. It is nevertheless possible to use whole tumor cells as a source of tumor Ags. We describe the purification of large numbers of human NHL B cells directly from lymph node or spleen biopsies, and different preparations allowing their use in a clinical setting. METHODS: The purification procedure consists of the negative selection of tumor B cells: cells to be eliminated are opsonized by CD2 Abs, and then coupled to magnetic beads for separation by the Isolex 300 magnetic separator. RESULTS: The mean yield of the purification was 74% for CD19+ cells, with a mean purity of 87%, dependent on the initial fraction of tumor cells in the biopsy. Using this procedure, a large number of purified tumor cells can be recovered from a biopsy in sterile conditions. We also describe treatments of B cells that can enhance their uptake by APCs, a critical step in anti-tumor immunotherapy strategies. Cells were opsonized by rituximab, or induced in apoptosis by irradiation, or necrosis by heating. Cell lysates were directly prepared from purified tumor cells. DISCUSSION: These procedures were reproducible on every lymphoma cell, and treated cells were phagocytosed by APCs. The methodology described here allows the evaluation of the immunological potential of apoptotic, necrotic, opsonized lymphoma cells, or their lysates, in a clinical setting.

Mechanisms of action of extracorporeal photochemotherapy in the control of GVHD: involvement of dendritic cells.

Despite the fact that extracorporal photochemotherapy (ECP) is now broadly used for the treatment of graft versus host disease or T-cell lymphomas, the mechanisms of its action remain enigmatic. This work provides a synthesis of the main results suggesting the initiation by ECP of an immune reaction responsible for the down modulation of pathogenic T-cell functions, with a special focus on the role of dendritic cells in this phenomenon.

Preparation of autologous leukemia and lymphoma vaccines expressing alpha-gal epitopes.

This study describes a novel method for increasing the immunogenicity of autologous tumor vaccines in leukemia and lymphoma patients by exploiting the natural anti-Gal antibody for in situ targeting of the vaccinating cells to antigen-presenting cells (APCs). Incubation of leukemia or lymphoma cells with neuraminidase and recombinant alpha 1,3-galactosyltransferase results in the synthesis of many alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on their cell membranes. Vaccination with such processed tumor cells results in the binding of the natural anti-Gal immunoglobulin G (IgG) antibody to these epitopes and opsonization of these cells for effective phagocytosis by APCs, such as dendritic cells and macrophages. These APCs may transport the vaccine to adjacent draining lymph nodes for subsequent effective processing and presentation of tumor-associated antigens (TAA) peptides to activate TAA-specific helper and cytotoxic T cells. Once the TAA-specific cytotoxic T cells are activated, they can leave the lymph node, circulate in the body, and seek metastatic cells expressing TAA to destroy them. Alternatively, activated helper T cells may provide the help required for B cells to produce antibodies to TAA on the leukemia or lymphoma cells. Because every patient receives his or her own TAA within the vaccinating cells, such vaccines are customized for the patient. These autologous tumor vaccines may be used as an adjuvant treatment that complements currently used treatment regimens by providing the immune system with an additional opportunity to be exposed effectively to autologous TAA.

Identification of a leukemic counterpart of the plasmacytoid dendritic cells.

This work aims to demonstrate that CD4(+)CD56(+) malignancies arise from transformed cells of the lymphoid-related plasmacytoid dendritic cell (pDC) subset. The analysis of malignant cells from 7 patients shows that in all cases, like pDCs, leukemic cells are negative for lineage markers CD3, CD19, CD13, CD33, and CD11c but express high levels of interleukin-3 receptor alpha chain (IL-3Ralpha), HLA-DR, and CD45RA. Tumor cells produce interferon-alpha in response to influenza virus, while upon maturation with IL-3 they become a powerful inducer of naive CD4(+) T-cell proliferation and promote their T-helper 2 polarization. As pDCs, leukemic cells also express pre-Talpha and lambda-like 14.1 transcripts, arguing in favor of a lymphoid origin. In addition, malignant cells express significant levels of CD56 and granzyme B. Overall, those observations suggest that CD4(+)CD56(+) leukemic cells could represent the malignant counterpart of pDCs, both of which are closely related to B, T, and NK cells.

Multiple co-stimulatory signals are required for triggering proliferation of T cells from human secondary lymphoid tissue.

Vaccine-based therapies are being developed for a variety of cancers and their efficacy will be determined by their ability to stimulate T cells in the secondary lymphoid tissue. We found that T cells isolated from human secondary lymphoid organs (LT-T), in contrast to peripheral blood T cells (PB-T) are hyporesponsive to cross-linked anti-CD3 mAb (CD3c) even in the presence of exogenous IL-2. Using mAb to trigger CD2 and CD28 co-stimulatory molecules, we found that such dual co-stimulation of LT-T induces profound and sustained responses including CD25 expression, IL-2 secretion and proliferation. Different levels of co-stimulation produced a hierarchical pattern of responses in LT-T, which correlated with the degree of CD3-TCR down-regulation. Mature antigen-presenting cells (APC) restored the capacity of LT-T to proliferate to stimulation of the CD3-TCR complex. Blocking studies demonstrated that optimal proliferation was critically dependent on co-stimulation via CD2 and CD28 engaged by their ligands on the APC. Therefore, LT-T have increased co-stimulatory requirements as compared to PB-T, i.e. multiple co-stimulatory signals coupled to CD3-TCR triggering. Furthermore, LT-T were found to be dependent on APC for survival, in contrast to PB-T. Clearly, LT-T do not behave in a comparable way to PB-T and in vitro experiments assessing novel cancer vaccines should therefore use LT-T as the most appropriate population of responder T cells.

Correlation between apoptosis microarray gene expression profiling and histopathological lymph node lesions.

AIMS: Microarray technology has recently led to the identification of molecular prognostic subgroups in non-Hodgkin's lymphomas. To determine the usefulness of ready made macroarrays as routine diagnostic tools in haematopathology, lymph node biopsies were analysed using a cDNA macroarray containing genes involved in apoptosis, including caspases. METHODS: Nine biopsy specimens were analysed using total frozen tissues: four samples of B cell follicular lymphoma, two of B cell diffuse large cell lymphoma, and three of non-neoplastic lymph nodes from benign lymphadenitis. Nine cell populations were sorted from fresh tissues: malignant B cells from two patients with follicular lymphoma and two with diffuse large cell lymphoma, reactive B cells from two benign lymph nodes, reactive T cells from one benign lymph node, and virgin (mantle zone) B cells and germinal centre B cells from benign tonsils. Immunohistochemistry (IHC) on paraffin wax sections was performed for the localisation of caspases 2, 3, 4, 7, 8, and 9. RESULTS: In the clustered array data, sorted cells from samples sharing common histological lesions were grouped together, whereas the array/histology correlation was less satisfactory for tissues. The expression profiles of both the array and IHC methods correlated for most caspases and samples. CONCLUSIONS: Variations in array profiles of sorted cell populations can be associated with specific histological features, suggesting a possible diagnostic application of ready made apoptosis macroarrays in haematopathology.

Differentiation of antigen-presenting cells (dendritic cells and macrophages) for therapeutic application in patients with lymphoma.

The recent clinical trial in lymphoma using tumor antigen-loaded DCs (Hsu et al, Nature Med 1996; 2: 52) demonstrates the efficiency of the use of professional antigen presenting cells (APCs) for taking up, processing and presenting tumor protein in a vaccine strategy in cancer. However, the production of large quantities of clinical grade APCs remains to be resolved. Here, we describe that both dendritic cells (DCs) and macrophages (MOs) can be efficiently differentiated in large numbers from lymphoma patients in spite of their disease and previous therapy. These cells were produced using the VAC and MAK cell processors according to standard operating procedures. DCs and MOs were differentiated from circulating monocytes in gas permeable hydrophobic bags, with 2% autologous serum and in the presence of GM-CSF and IL-13 or GM-CSF alone, respectively. DCs and MOs were then purified by counter flow centrifugation. Phenotypic, morphological and functional analysis showed that cells differentiated from patients with lymphoma present quite similar features to DCs and MOs produced from monocytes of healthy donors. Moreover, we show that MOs, when combined with CD20 antibody (Rituximab), can efficiently engulf tumor cells and propose that a such combination could be used for initiating a clinical trial in lymphoma. Thus, the possibility of producing functional DC and MOs in large amounts in conditions compatible with therapeutic application will allow the development of new immune strategies to eradicate lymphoma.

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-Hodgkin's 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.

Expression of gene MAGE-A4 in Reed-Sternberg cells.

Genes of the MAGE-A family are expressed in several types of solid tumors but are silent in normal tissues with the exception of male germline cells, which do not carry HLA molecules.Therefore, peptides encoded by MAGE-A genes are strictly tumor-specific antigens that can be recognized by CTL and constitute promising targets for immunotherapy. The expression of 6 genes of the MAGE-A family was tested with reverse transcriptase-polymerase chain reaction in lymphoma samples. Among 38 samples of non-Hodgkin lymphoma, 1 anaplastic large cell lymphoma expressed genes MAGE-A1, -A2, -A3, -A4, and -A12, and 1 lymphoepithelioid T-cell lymphoma expressed gene MAGE-A4. Five of 18 samples (28%) from patients with Hodgkin disease expressed gene MAGE-A4. In tissue sections, staining by a monoclonal antibody that recognizes the MAGE-A4 protein was observed in 11 of 53 samples (21%) from patients with Hodgkin disease. In the positive samples, the Reed-Sternberg cells were strongly stained whereas the surrounding cells were not. These results indicate that Hodgkin disease may be a target for specific immunotherapy involving MAGE-A4 antigens.

Resistance to CD95-mediated apoptosis through constitutive c-FLIP expression in a non-Hodgkin's lymphoma B cell line.

CD95 (Fas/Apo-1) is a transmembrane molecule that induces apoptosis and plays a central role in the regulation of the immune response. The present study describes two new B lymphoid cell lines, B593 and BR97, derived from non-Hodgkin's lymphoma, which differ in susceptibility to CD95-mediated apoptosis. While B593 cells are sensitive to CD95mediated apoptosis, BR97 cells are completely resistant. Activation of caspase-8 and caspase-3 proteases plays an important role in the CD95 signalling pathway. CD95 stimulation induced caspase-8 and caspase-3 activation in B593, but not in BR97 cells. However, activation of both caspase-8 and caspase-3 was achieved in BR97 cells treated with staurosporine. Furthermore, protein synthesis inhibition by cycloheximide restored sensitivity to CD95-mediated apoptosis and allowed activation of both caspase-8 and caspase-3 in BR97 cells. These results indicate that, in BR97 cells, both caspases are functional and suggest that CD95-apoptosis resistance may result from the presence of inhibitory factor(s). Constitutive high level expression of the apoptotic inhibitor c-FLIP was observed in the CD95-resistant BR97 cell line compared to B593. Moreover, downregulation of c-FLIP expression level by protein synthesis inhibition strictly correlated with restored sensitivity to CD95-mediated apoptosis in BR97 cells. Furthermore, we demonstrate that c-FLIP is recruited to the CD95 DISC in BR97 cells together with caspase-8 and FADD. The data presented in this study strongly suggests that, in a B-NHL-derived cell line, resistance to CD95-mediated apoptosis results from endogenous high level expression of apoptotic inhibitor c-FLIP.