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.

Characterization of a receptor for interleukin 5 on human eosinophils: variable expression and induction by granulocyte/macrophage colony-stimulating factor.

Interleukin 5 (IL-5) acts on eosinophil differentiation and activation, suggesting the existence of a membrane receptor for IL-5 on eosinophils. Here, we report that 125I-labeled recombinant human IL-5 bound, at 4 degrees C, to high affinity receptors on human eosinophils. The association constant was higher for hypodense eosinophils (1.93 x 10(9) M-1) than for normodense cells (0.39 x 10(9) M-1), with a closely related number of receptor sites per cell. No specific binding occurred on neutrophils. The specific binding of IL-5 was induced by overnight incubation at 37 degrees C of human eosinophils with granulocyte/macrophage (GM)-CSF. The levels of increase were significantly higher for normodense than for hypodense eosinophils, suggesting a previous in vivo activation of the later subpopulation by GM-CSF. IL-3 was ineffective by itself but synergistically enhanced the effect of GM-CSF. Specificity studies showed that the binding of 125I-labeled IL-5 was inhibited by IL-5, but not by other cytokines, on human eosinophils. These results show the existence of a specific binding site for IL-5 on human eosinophils with a variable affinity on eosinophil hypodense or normodense subpopulations, as previously reported for other membrane receptors.

Interleukin 5 messenger RNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease.

Interleukin 5 (IL-5), the major factor involved in eosinophil differentiation, is produced by T cells or mast cells. In the present study, we found that eosinophils infiltrating the mucosa of four patients with active coeliac disease also express the IL-5 mRNA. No positive signal was obtained in normal duodenum tissues and in the cell infiltrate from patients submitted to gluten restriction. The identification of labeled mucosal cells as eosinophils relied on their typical morphology. Moreover, highly purified blood eosinophils from three out of four patients with eosinophilia were also strongly labeled with the IL-5 antisense but not with the corresponding sense probe. Together, these results suggest that eosinophils have the capacity to synthesize IL-5, which could contribute to paracrine interactions with T and B cells and, in autocrine fashion, locally participate, through binding to the IL-5 receptor, to eosinophil differentiation and activation. These data might have implications not only in the pathology of coeliac disease but also in other diseases associated with eosinophil infiltration.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Signal transduction via human leucocyte antigen class II molecules distinguishes between cord blood, normal, and malignant adult B lymphocytes.

Cord blood is increasingly used for hematopoietic stem cell transplantation since less severe graft-versus-host disease has been reported leading to the notion that cord blood is "naive." Human leucocyte antigen (HLA) class II molecules are expressed throughout B lymphocyte ontogeny (except the plasmocytes), are responsible for antigen presentation, and can also transmit signals. Cord blood B stimulate an allogeneic response, and this property is believed to indicate the presence of a class II-associated peptide. In this study we examined the capacity of cord blood B to transmit signals via HLA-DR. Activation and relocalization of protein kinase C (PKC) isoenzymes alpha and betaII was detected along with tyrosine kinase activation and proliferation. However, in contrast to resting adult B, generation of an intracellular calcium ([Ca++]i) flux and rapid aggregation were not detected. To address the question of whether or not HLA-DR signals throughout B lymphocyte ontogeny, we extended this study to include malignant adult B (B chronic lymphocytic leukemia [B-CLL], B mantle cell lymphoma, and B large cell leukemia). Tyrosine kinase activation and proliferation were observed in all these cell populations, albeit in the absence of [Ca++]i flux or an increase in PKC. HLA-DR therefore transmits signals throughout B lymphocyte ontogeny, although different signaling pathways are initiated in adult vs. fetal vs. malignant B. The lack of intracellular [Ca++]i flux in both cord blood and malignant B lymphocytes may represent a feature of HLA class II signaling at a particular stage of differentiation, although the downregulation of PKC clearly distinguishes between cord blood B and B-CLL.

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.

The interleukin 2 receptor in the hypereosinophilic syndrome.

The hypereosinophilic syndrome (HES) has been previously described as a clinicobiological entity characterized by a blood eosinophil count of over 1.5 x 10(9)/L of unknown cause associated with several clinical complications. In reality, HES is a heterogeneous group of diseases with variable and unpredictable progress in visceral lesions, thought to be related to the deleterious effects of tissue eosinophil infiltration. Various criteria for discrimination between benign and severe forms of HES have been described. These previous retrospective clinical investigations, using biological and clinical markers, have defined different stages of HES. It appears more relevant, however, to consider elements of disease activity by studying mechanisms of induction of persistent hypereosinophilia. The T-cell dependence of blood eosinophilia has led us to evaluate various markers of T-cell activation in particular. In the present review, we report previous results and perspectives suggested by the study of the interleukin 2 receptor in HES.

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.

Induction of CD23, CD25 and CD4 expression on an eosinophilic cell line (EoL-3) by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5).

The effects of IL-3, GM-CSF and IL-5 on the expression of CD23 (Fc epsilon RII), CD25 (IL-2R/p55) and CD4 on an eosinophilic cell line (EoL-3) were investigated by flow cytometry. A separate incubation with IL-3, GM-CSF or IL-5 alone, did not induce the expression of CD23, CD25, or CD4. However, a sequential incubation with IL-3 for 6 days, then with IL-3 and GM-CSF for the following 6 days, induced a significant expression of CD23 and CD25. After a further incubation for 6 days with IL-3, GM-CSF and IL-5, CD4 was then expressed, while CD23 and CD25 expression still increased. The kinetics of expression of CR3/CD11b were parallel to that of CD23, but the expression of the transferrin receptor (CD71) remained negative. Northern blot analysis revealed the presence of mRNA encoding CD23, CD25 and CD4 in EoL-3 stimulated by IL-3, GM-CSF and IL-5. Culture with GM-CSF induced the binding of radiolabeled IL-5 to EoL-3 cells, with an increased affinity after incubation with IL-3, GM-CSF and IL-5. These data indicate that IL-3, GM-CSF and IL-5, might be involved in the expression of functional markers on eosinophil membrane.