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.

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.

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.

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.

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.

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.

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.

Malignant B lymphocytes from non-Hodgkin's lymphoma induce allogeneic proliferative and cytotoxic T cell responses in primary mixed lymphocyte cultures: an important role of co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) in stimulation by tumor cells.

We analyzed the stimulating capacities of malignant B cells from non-Hodgkin's lymphomas (NHL) to induce an allogeneic response in primary mixed lymphocyte reaction (MLR). T cells purified from a single healthy donor (KS) were used to compare the responses induced by either malignant or hyperplastic cells. Malignant B cells induced strong proliferation of KS cells independently of their level of expression of adhesion molecules. The KS cells after MLR were predominantly CD3+, CD25+, HLA-DR+, Ki67+ and CD45RO+ T cells, and the CD4/CD8 ratio was heterogeneous (from 0.8 to 2.7). To investigate the role of co-stimulatory molecules CD80 and CD86 for the stimulatory capacities of B cells, the expression of both molecules was analyzed before and during the MLR. Most fresh malignant B cells were negative for CD80 and CD86, whereas co-cultured B cells expressed high levels of both molecules. This expression was crucial for T cell proliferation, since monoclonal antibodies directed against CD80 and CD86 completely abrogated the MLR. We also report that KS responding cells at the end of co-culture were able to lyse fresh B cells used as stimulator cells to different extents (from 10 to 51%), and the level of lysis was enhanced after PMA activation of the target cells. Inhibition experiments using CD8 and CD4 mAb showed that effector cells were mainly CD8+. This report is the first to describe the accessory function of human malignant B cells from NHL and their sensitivity to lysis mediated by CD8+ T cells, and suggests new strategies for the development of antitumor immunity in NHL.

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.

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.