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.

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.

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.

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.

Quantitative analysis detects ubiquitous expression of apoptotic regulators in B cell non-Hodgkin's lymphomas.

To determine whether the expression levels of Bcl-2 family apoptotic regulators are correlated with the histopathological heterogeneity of B cell non-Hodgkin's lymphomas (NHL), we quantified their expression in malignant B cell populations isolated from 33 biopsy samples, including small lymphocytic lymphoma (SLL, n = 9), mantle cell lymphoma (MCL, n = 8), follicular lymphoma (FL, n = 8), and diffuse large cell lymphoma (DLCL, n = 8). Normal B cells purified from reactive lymph nodes and tonsil (n = 3) were used as controls. Cell lysates were analyzed by Western blotting, and signals quantified by densitometry. Expression of Bcl-2 and its homologues, Bcl-xL, Bcl-xS, Bax, Bad, Bak and Bag-1, was detected in all NHL cases, with wide variations between histological subtypes and within each subtype. Statistically significant differences were: (1) a higher level of Bad expression in DLCL compared to FL and MCL; (2) a lower level of Bak expression in FL compared to DLCL, SLL and MCL; and (3) a higher Bag-1 expression level in FL compared to SLL. When compared to NHL cells, normal B cells showed a higher level of Bax expression, and a lower level of Bcl-xL expression. Thus, quantitative analysis shows ubiquitous expression of Bcl-2 family proteins in normal and neoplastic B cells; the variations in expression levels may contribute to both the B-NHL clinicopathological diversity and the different apoptotic sensitivities of normal B cells vs B-NHL cells.

Quantification of cellular adhesion molecules on malignant B cells from non-Hodgkin's lymphoma.

The expression of five cellular adhesion molecules (CAMs), CD54, CD58, CD11a, CD29 and CD49d, was studied in 113 B cell non-Hodgkin's lymphomas (NHL) and in normal B cells from 12 control lymph nodes. Rather than reporting the percentage of positive cells, which does not discriminate between NHL subtypes, we quantified the intensity of CAM expression using flow cytometry. Apart from CD49d the expression of all these CAMs was statistically different among the NHL subtypes as defined by the REAL classification. Low grade NHL-small lymphocytic, follicular and mantle cell lymphoma--which are derived from quiescent cells and show an indolent disease course, expressed low levels of CAMs. Conversely, high grade NHL-diffuse large cell lymphoma--which are derived from proliferating cells and are clinically aggressive, expressed high levels of CAMs. These results indicate that in malignant NHL B cell tumour growth and clinical aggressiveness may be related to the adhesive capacities of the tumour 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-Hodgkin's 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.

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-Hodgkin's 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.

Tumor B cells from non-Hodgkin's lymphoma are resistant to CD95 (Fas/Apo-1)-mediated apoptosis.

Apoptosis mediated by the CD95 (Fas/Apo-1) molecule plays a crucial role in the regulation of the B-cell immune response. In this study, we examined the function of the CD95 antigen in B-cell-derived non-Hodgkin's lymphoma (NHL), a malignant disease of mature B cells. Membrane CD95 molecules were found to be constitutively expressed in a large number of NHL, including mantle cell (MCL, n = 10), lymphocytic (LCL, n = 10), follicular (FL, n = 11), and diffuse large cell lymphoma (DLCL, n = 9) with, however, different levels of intensity. Indeed, the levels of CD95 were low in MCL and LCL as compared with FL and DLCL. However, regardless of the intensity of expression, CD95 triggering with anti-CD95 monoclonal antibody (MoAb) did not induce apoptosis of lymphoma B cells, while these cells underwent apoptosis after irradiation or staurosporine treatment. Further experiments were then performed to address whether apoptosis could be restored by B-cell activation via CD40 cross-linking. We showed that CD40 engagement in the presence of interleukin (IL)-4 was more effective than CD40 engagement alone in upregulating the CD95 antigen and induced CD95-mediated cell death in nontumoral B cells. Concerning malignant B cells, CD40 ligation in the presence of IL-4 strongly increased CD95 expression, but did not markedly increase CD95-induced apoptosis. Furthermore, using cytotoxic T cells, we showed that CD95L was also ineffective in inducing apoptosis in lymphoma B cells, whereas these cells were killed by the perforin pathway. Our findings suggest that the CD95-mediated cell death pathway is altered in malignant cells from the NHL we tested. This could be a mechanism allowing lymphoma B cells to escape from immune regulation.

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-Hodgkin's 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.