Acute Lymphoblastic Leukemia research paper Thymic epithelial cells promote survival of human T-cell acute lymphoblastic leukemia blasts: the role of interleukin-7 MARIA T. SCUPOLI, FABRIZIO VINANTE, MAURO KRAMPERA, CARLO VINCENZI, GIANPAOLO NADALI, FRANCESCA ZAMPIERI, MARY A. RITTER, EFREM EREN, FRANCESCO SANTINI, GIOVANNI PIZZOLO Background and Objectives. T-cell lymphoblastic -cell acute lymphoblastic leukemia (T-ALL) is a leukemia (T-ALL) cells originate within the thymus from malignant disease resulting from the clonal prolif- the clonal expansion of T cell precursors. Among thymic Teration of T lymphoid precursors. It accounts for stromal elements, epithelial cells (TEC) are known to exert about 15% of all ALL cases in children and 20-25% in a dominant inductive role in survival and maturation of adults.1,2 T-ALL is thought to originate inside the thy- normal, immature T-cells. In this study we explored the mus and leukemic cells express phenotypic features cor- possible effect of TEC on T-ALL cell survival and analyzed responding to distinct maturational stages of thymo- the role of interleukin-7 (IL-7) within the microenviron- cyte development: early (stage I), intermediate (stage II), ment generated by T-ALL-TEC interactions. or late (stage III).2,3 Design and Methods. T-ALL blasts derived from 10 The thymus is the main site where bone marrow adult patients were cultured with TEC obtained from (BM)-derived stem cells differentiate into mature, human normal thymuses. The level of blast apoptosis was immunocompetent T lymphocytes.4,5 The internal stro- measured by annexin V-propidium iodide co-staining and mal framework of the thymus is composed of epithelial flow cytometry. The proliferative response of leukemic cells cells, interspersed with BM-derived macrophages and to interaction with TEC was evaluated by thymidine incor- dendritic cells, surrounded by extracellular matrix to poration at various time intervals of culture. To assess the form an intralobular meshwork filled with developing T role of IL-7, lympho-epithelial co-cultures were carried lymphocytes.6-8 The thymic epithelium is composed of out in the presence of anti-IL-7 or anti IL-7R blocking antibodies and the level of apoptosis of T-ALL blasts was multiple, distinct cellular subsets, characterized by analyzed. tonofilaments and interconnected by desmosomes. Results. When T-ALL cells were cultured in the pres- Three broad classes of epithelium have been identified ence of TEC monolayers, the percentage of viable cells by using monoclonal antibodies to antigen determi- increased significantly and this survival was sustained nants: subcapsulae/subtrabeculae/perivascular, cortex, with time in culture. In addition, the interaction with TEC and medulla.6 Although distinct functions of the differ- induced a considerable proliferative response in T-ALL ent subsets have not been fully elucidated, it is known cells (15-fold greater than that of the control cells after that thymic epithelial cells (TEC) exert a pivotal role in 7 days of culture). The presence of IL-7 or IL-7R blocking the homing, intrathymic migration, and differentiation of antibodies in lympho-epithelial co-cultures consistently thymocytes through the release of cytokines, the secre- reduced the TEC-mediated apoptosis inhibition in T-ALL tion of extracellular matrix components, and the estab- blasts (70% decrease). lishment of adhesive interactions.9-13 Furthermore, in vit- Interpretation and Conclusions. These results point ro experiments using co-cultures between thymocytes to the role of thymic epithelium in the regulation of T blast and TEC or mouse fetal thymic organ culture (FTOC), survival. In addition, they show that interaction between indicate that adhesive interactions with TEC or with IL-7 and its receptor has the major role in modulating T- factors secreted by TEC can protect thymocytes from ALL survival within the microenvironment generated by apoptosis.14,15 A recent report indicates that mouse FTOC the T-ALL/TEC interaction. can induce the proliferation of blasts from T-ALL patients, thus suggesting that thymic stromal elements Key words: human, thymus, T-ALL, TEC, interleukin-7. may also have a role in regulating the growth of malig- nant thymocytes.16 Haematologica 2003; 88:1229-1237 http://www.haematologica.org/2003_11/1229.htm Interleukin-7 (IL-7) has a critical, non-redundant role in normal T-cell development. IL-7 is secreted by thy- ©2003, Ferrata Storti Foundation mus and BM stromal cells and exerts its activity by sig- naling through a receptor complex consisting of the IL- From the Department of Clinical and Experimental Medicine, Section of 7Rα chain and γc. Mice deficient in IL-7 have a drasti- Haematology, University of Verona (MTS, FV, MK, CV, GN, FZ, GP); Division of cally reduced number of thymocytes and a defect in Cardiac Surgery, Azienda Ospedaliera di Verona, Verona, Italy (EE); Dept. of Immunology, Faculty of Medicine, Imperial College, London, the developmental transition from immature to T-cell United Kingdom (EE, MAR). committed thymocytes.17 Similarly, mice deficient in IL- Correspondence: Maria Teresa Scupoli, Dipartimento di Medicina Clinica e 7R show an early defect in lymphopoiesis, and the few Sperimentale, Sezione di Ematologia, Università di Verona, Policlinico G.B. mature T cells which do develop are functionally Rossi, ple. L.Scuro 10, 37134 Verona, Italy. impaired.18 The critical role of IL-7 for early T-cell devel- E-mail: [email protected] haematologica/journal of hematology vol. 88(11):november 2003 1229 M. T. Scrupoli et al. opment was also demonstrated in chimeric mary cultures were detached by trypsin-EDTA human-mouse thymus organ culture models in treatment, plated on a 3T3-J2 feeder layer and which antibodies blocking IL-7 and IL-7R were expanded to confluent secondary cultures in used.19 Furthermore, IL-7 is involved in leukemo- growth medium. TEC destined to co-culture exper- genesis as IL-7 transgenic mice develop lymphoid iments were derived from secondary cultures, tumors.20 Different studies have indicated a role already devoid of 3T3-J2 cells, and grown to con- for IL-7 in regulating survival and cell cycling of fluence. Media were purchased from Seromed blasts in T-ALL patients, thus suggesting that IL-7 (Berlin, Germany), EGF from Austral Biological (San may regulate the expansion of malignant cells.21-27 Ramon, CA, USA), and supplements from Sigma- However, in FTOC systems the growth of T-ALL cells Aldrich (Milan, Italy). appears to be independent from IL-7.16 Thus, the The HeLa human epithelial-like cell line was cul- putative role of IL-7 as a survival factor for T-ALL tured in DMEM (GIBCO), 10% FCS, L-glutamine, blasts in the thymus has not yet been fully eluci- and antibiotics. dated. In this report, we show that TEC reduce sponta- Co-cultures and antibody treatment neous apoptosis and induce proliferation in cells TEC or HeLa cells destined to co-cultures were from T-ALL patients. Furthermore, the functional grown to a confluent state. For proliferation assays, blockage of IL-7 or IL-7R reduces TEC-mediated epithelial monolayers were sublethally irradiated apoptosis inhibition in T-ALL. We propose that TEC 24 hours before use whereas non-irradiated TEC have a functional role in modulating the survival of were used for apoptosis analysis of blasts. Imme- T-ALL blasts by a mechanism mainly dependent on diately before experiments, the medium from the interaction between IL-7 and its receptor. epithelial monolayers was removed and the cells washed extensively. T-ALL blasts were added to Design and Methods epithelial monolayers at a 10:1 blast:epithelial-cell ratio and cultured in a humidified atmosphere of Cells 5% CO2, in medium composed of DMEM and Ham’s Peripheral blood samples were collected, after F12 medium (3:1 mixture), 10% FCS, 5 µg/mL informed consent, from 10 adult patients with insulin, 5 µg/mL transferrin, 0.18 µM adenine, 4 newly diagnosed T-ALL. Mononuclear cells were mM L- glutamine, and 50 IU/mL penicillin-strep- isolated by Lymphoprep density gradient centrifu- tomycin. Control T-ALL blasts were cultured in the gation (Nicomed, Oslo, Norway). In two co-culture same conditions but in the absence of epithelial experiments (cases 1 and 3), cells were used imme- monolayers. At the end of co-culture, cells were diately after preparation whereas cryopreserved harvested by vigorous pipetting. The lack of resid- samples were employed, immediately after thaw- ual cells in the wells was assessed by phase con- ing, in the other cultures. Comparative experiments trast microscopy. The T-cell nature of the cells har- showed no significant differences between results vested from co-cultures was assessed by immuno- obtained from fresh or thawed samples (data not fluorescence with anti-CD5 monoclonal antibody shown). Before use, the cells’ viability consistently and flow cytometry (data not shown). exceeded 90% in each sample, as assessed by pro- For blocking experiments, neutralizing concen- pidium-iodide (PI) dye exclusion. trations (10 µg/mL) of antibodies recognizing func- Thymic epithelial cell cultures were derived, after tional epitopes of IL-7 (rabbit polyclonal, Biosource informed consent, from normal thymuses of chil- International, Camarillo, CA, USA) or IL-7R (clone dren (< 5 years of age) undergoing cardiac surgery, R34.34, Instrumentation Laboratory, Milan, Italy) as previously described.28,29 Briefly, thymus speci- were added to the T-ALL/TEC co-cultures at the mens were minced and treated with a 0.05% start of the assay. trypsin-0.01% EDTA solution at 37°C for 3 hours. Cells were collected every 30 minutes, pooled, plat- Immunophenotype analysis ed onto lethally irradiated 3T3-J2 cells30 (kindly Immunophenotype analysis of T-ALL blasts and provided by Dr H Green, Harvard Medical School, TEC was performed by direct immunofluorescence Boston, MA, USA) at 2.5×104/cm2, and cultured in and flow cytometry with a FACScalibur instrument a humidified atmosphere of 5% CO2, in growth (Becton-Dickinson, San José, CA, USA).