Jean-Claude Bensa

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.

Quality control for the validation of extracorporeal photopheresis process using the Vilbert-Lourmat UV-A irradiation's system

In agreement with good practices for therapeutic use of human cells, quality control has to be performed to valid the process of extracorporeal photopheresis (ECP) with the Vilbert-Lourmat system. Since no protocol exists, we evaluated a technique based on the measurement of the inhibition of mitogen (PHA, Con-A, OKT3)-induced proliferation, in 164 procedures from 16 patients. Whatever the pathology, we observed a high proliferation rate in most samples, and we obtained over 90% ECP-induced inhibition in as many as 94% of the cases. Since this approach proved to be relevant regarding our objective, a protocol for the ECP process validation is proposed.

Autotumour reactive T-cell clones among tumour-infiltrating T lymphocytes in B-cell non-Hodgkin's lymphomas.

Summary. Seventy‐three T‐cell clones (TCC) were established from tumour‐infiltrating lymphocytes‐T (TIL‐T) derived from lymph nodes involved by B‐cell non‐Hodgkins lymphomas (B‐NHL) in nine patients with different histological subtypes and clinical stages. 40 TCC (55%) expressed the CD25 Ag and were also able to proliferate in the presence of irradiated autologous B‐NHL cells. Among them, 23 autotumour (AuTu) proliferative TCC were found not to proliferate to autologous EBV‐transformed B‐cell lines, indicating that the proliferative reactivity of these TCC was preferentially directed at autologous B‐NHL cells. Tested against autologous B‐NHL cells, only three AuTu prolifera‐ tive TCC (CD8 +) showed a significant level of cytotoxicity (specific lysis > 15%). In blocking experiments, the AuTu proliferative reactivity of three TCC from one patient was strongly inhibited by anti‐DR and anti‐DQ mAbs, whereas that of three TCC from another patient was not affected by either anti‐MHC class I or class II (DR., DP, DQ) mAbs. These findings suggest that the recognition of autologous B‐NHL cells by AuTu proliferative TCC may occur through MHC‐restricted as well as MHC‐unrestricted mechanisms.

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.

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.

Immunomonitoring of graft-versus-host minor histocompatibility antigen correlates with graft-versus-host disease and absence of relapse after graft

BACKGROUND: After HLA‐identical hematopoietic stem cell transplantation, minor histocompatibility (mH) antigen alloreactivity plays a dominant role in the development of graft‐versus‐host disease (GVHD) and graft versus leukemia (GVL).

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.

Quantitating Effector and Regulatory T Lymphocytes in Immune Responses by Limiting Dilution Analysis Modeling

Although there is currently no doubt that regulatory lymphocytes represent a master player in the immune system, a major unresolved problem is the accurate quantitation of these cells among unfractionated cell populations. This difficulty mainly arises because there are no specific immunophenotypic markers that can reliably discriminate between effector and regulatory lymphocytes. To face this problem, we have developed computational models of limiting dilution analyses addressing the question of the accurate estimation of the frequencies of effector and regulatory cells functionally engaged in an immune response. A set of generic equations were provided to form a framework for modeling limiting dilution data, enabling discrimination between qualitatively different models of suppression. These models include either one or two subpopulations of regulatory cells, featured by either low or potent regulatory activity. The potential of this modeling approach was illustrated by the accurate determination of the frequencies of effector and regulatory T lymphocytes in one real limiting dilution experiment of CD4+CD25+ T lymphocytes performed in the context of an allogeneic response in the human system. The crucial advantage of the limiting dilution method over the “static, phenotype-based” method is the dynamic evaluation of effector and regulatory T cell biology through their actual functional activity.