ORIGINAL ARTICLE Alloreaction increases or restores CD40, CD54, and/or HLA molecule expression in acute myelogenous leukemia blasts, through secretion of inflammatory cytokines: dominant role for TNFb, in concert with IFNc A Eljaafari 1,2 , J Van Snick 3 , A Voisin 1 , F Cormont 3 , A Farre 1 , J Bienvenu 4 , J Bernaud 5 , D Rigal 5 and X Thomas 6 1 Cell Therapy Research Department, EFS-Rhone-Alpes, Lyon, France; 2 Immunogenetis Unit, HCL/BioMerieux, Hopital E. Herriot, Lyon, France; 3 Ludwig Institute for Cancer Research and Experimental Medicine Unit, Catholic University of Louvin, Brussels, Belgium; 4 Immunology Department, CHLS, Lyon, France; 5 Immunology Department, EFS-Rhone-Alpes, Lyon, France and 6 Hematology Department, Hopital E. Herriot, Lyon, France We have previously reported that alloreaction can lead to activation of dendritic cells through secretion of inflammatory cytokines. Here, we addressed whether alloreaction-derived cytokines may also lead to acute myelogenous leukemia (AML) blast differentiation. With this aim, supernatant (sn) harvested from major or minor histocompatibility antigen-mismatched mixed lymphocyte reaction (MLR) were used to culture French American Bristish (FAB) type M4 or M5 AML blasts. Our results showed that the secreted factors induced upregulation of CD40, CD54, and/or HLA molecules in AML blasts. Protein fraction- ation, blockade experiments and exogenous cytokine reconsti- tution demonstrated the involvement of TNF in the upregulation of CD54, CD40 and HLA-class II molecules, and of IFNc in the increase of HLA-class I and class II molecule expression. But, in line of its much higher levels of secretion, TNFb, rather than TNFa, was likely to play a preponderant role in AML blast differentiation. Moreover TNFb and IFNc were also likely to be involved in the AML blast differentiation-mediated by HLA- identical donor T-cell alloresponse against recipient AML blasts. In conclusion, we show herein that upon allogeneic reaction, TNFb secretion contributes, in concert with IFNc, to increase or restore surface molecules involved in AML blast interaction with T cells. Leukemia (2006) 20, 1992–2001. doi:10.1038/sj.leu.2404375; published online 14 September 2006 Keywords: acute myelogenous leukaemia; allogeneic reaction; blast differentiation; TNFb; IFNg Introduction Allogeneic bone marrow transplantation (BMT) is a therapeutic approach used to eradicate hematological malignancies, such as leukemias, 1–2 or even solid tumors. 3 However, this treatment is associated with severe side effects, the most serious of which is graft-versus-host-disease (GVHD). 4 At present, the understand- ing of the effector mechanisms that trigger GVHD implicates both cell-mediated cytotoxicity against host tissues and inflam- matory cytokines. Thus, the dissection of the patho-physiologi- cal mechanisms that are involved in GVHD demonstrates several sequential steps. 5–9 In the first phase, cytokines, such as TNFa, IL-1 and IL-6, are secreted during the conditioning regimen and contribute to activation of donor T cells. Subsequently, stimulation of donor T cells by host antigens in concert with the phase 1-secreted cytokines leads to production of cytokines, including, IL-2 and IFN-g, which amplify the allogeneic response and activate mononuclear phagocytes. The third phase involves secretion of inflammatory cytokines, such as IL-1 and TNFa, by these latter cells. These cytokines, in synergy with T and NK cell-mediated cytotoxicity, induce host tissue injury. Acute GVHD results in deleterious effects which target mostly skin, digestive tract, and liver. 10 Moreover, it has been reported in the latter disease stage, that release of lipopolysaccharide (LPS) by damaged epithelial tissues en- hances effector functions of mononuclear phagocytes (for review see reference 11,12 ). Prognosis would then depend on the disease stage. Like GVHD, graft-versus-leukemia (GVL) is known to principally result from cell-mediated cytotoxicity, but here the targets are leukemic cells, rather than host tissues. 13–16 Thus, because GVHD facilitates GVL and due to the knowledge that leukemic cells of myeloid origin with monocytic compo- nents can differentiate into potent antigen presenting cells (APC), like dendritic cells, depending on the cytokine environ- ment, 17–20 we questioned here whether secretion of cytokines during GVHD could enhance the antigen presenting capacity of leukemic cells. To test this hypothesis, we performed in vitro allogeneic mixed leukocyte cultures, harvested supernatant (mixed leukocyte reaction (MLR-Sn)) and used them to culture acute myeloid leukaemia (AML) blasts from 10 patients with monocytic components, or AML blast cell lines. Their differ- entiation was evaluated by analyzing their cell surface phenotype. Methods Medium and reagents RPMI 1640 (Life Technologies, Eggenstein, Germany) was supplemented with L-glutamin (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), NaHCO 3 (1.5 mg/ml) and 10% of pooled human AB heat inactivated serum. In other experiments X-VIVO 20 (Bio-Whittaker, Walkersville, MD, USA) was used instead of RPMI 1640. In the latter case, neither serum nor antibiotics, NaHCO 3 , nor L-glutamine were added. Patients Ten patients with newly diagnosed acute myeloid leukemia were studied. Diagnosis was based on May–Gru ¨ nwald–Giemsa stained bone marrow smears and cytochemistry according to the French-British-American-Group criteria. Morphology and karyotypic characteristics of patients are shown in the table below. All patients gave written informed consent to the study Received 15 February 2006; revised 17 July 2006; accepted 21 July 2006; published online 14 September 2006 Correspondence: Dr A Eljaafari, Immunogenomics Unit, HCL/Biomer- ieux, ERT 1041, Hopital E. Herriot, Pavillon P, 5 place d’Arsonval, 69003 Lyon, France. E-mails: eljaafari@aol.com, assia.eljaafari@chu-lyon.fr Leukemia (2006) 20, 1992–2001 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu