Haematologica 2004;89: supplement no. 6 - Supplements ...

164PostersPO-168TLR7 AND TLR9 LIGANDS EXHIBIT IMMUNOSTIMULATORYACTIVITIES ON CHRONIC LYMPHOCYTIC LEUKEMIA B CELLSFilì L,* Brugnolo F,* Sampognaro S,* Liotta F,* CosmiL,* Manuelli C, # Santini V, § Ciolli S, § Bosi A, § MaggiE,* Romagnani S,* Annunziato F,* Parronchi P**Dept of Internal Medicine, Section of Immunoallergology,# Dept. of Dermatological Sciences and § Dept.of Hematology, University of Florence, ItalyNatural and synthetic Toll-like Receptor (TLR) ligandscan activate cells of the innate immunity andsome of them also show the ability to modulateimmunocompetent cells. In particular, short-lenghtCpG-containing motif oligodeoxynucleotides (CpG-ODNs) can stimulate proliferation and differentiationof murine and human B cells. More recently, imidazoquinolineshave been demonstrated to stimulatenormal human B cells through a TLR7 (and 8)-mediatedsignaling. Chronic lymphocytic leukemia (B-CLL)cells represent a mature malignant counterpart of Bcells characterized by high apoptosis rate in vitro,unresponsiveness to ordinary agents promoting differentiationand proliferation. Limited data are knownon TLR in B-CLL cells. In our study, the analysis ofTLR1-10 by real-time PCR showed that highly purifiedCD5+ B-CLL cells expressed TLR7 and 9 whereasTLR8 and TLR1-5 were negative. We then comparedthe effects of TLR7-ligand R-848 (Resiquimod)with the phosphorothioate CpG ODNs DSP30 and2006 which bind to TLR9.For control, LPS and syntheticdouble strand RNA poly I: poly C were used. R-848 was able to induce a significant proliferation ofCD5+ leukemic B cells in almost all the analyzed B-CLL (90% cases) in a dose-curve manner with a similarrate of proliferation to CpG-ODNs. CFDA-SEanalysis confirmed that CD19+CD5+ cells wereresponsible for thymidine-uptake. The stimulatoryeffect was directed to B cells as the depletion of PDCsdid not affect the proliferative response. More interestingly,R-848 induced a strong up-regulation ofcostimulatory molecule expression (CD40, CD80,CD86) together with increased expression of CD20and MHC class II molecules and R-848 and CpG-ODNpre-incubated B cells also exhibited increased stimulatoryability to allogeneic T cells in MLR even atvery low E:R ratios (1:500). Finally, R-848 and, in amore limited way, CpG-ODNs were able to stimulatethe differentiation of B-CLL cells into actively IgMproducingcells. Taken together, our data indicate thatResiquimod is a potent immunomodulator to inducematuration of neoplastic B cells to stimulate an efficientT cell response in some haematologic malignancies.PO-169HIGH LEVELS OF ACTIVATED CASPASES IDENTIFY PATIENTSWITH INCREASED RISK OF DISEASE PROGRESSION IN B-CELLCHRONIC LYMPHOCYTIC LEUKEMIADel Poeta G, Del Principe MI, Luciano F,Irno Consalvo M, Mazzone C, Suppo G, Marini R,Maurillo L, Venditti A, Buccisano F, Bruno A,Piccioni D, Franchi A, Meoni G, Amadori SCattedra di Ematologia, Università “Tor Vergata”,Ospedale S. Eugenio, Rome, ItalyB-cell chronic lymphocytic leucemia (B-CLL) is adisease characterized by the progressive accumulationof tumor cells which do not proliferate rapidly,but fail to undergo death. Even though almost allperipheral blood B-cells are arrested in G0 phase ofthe cell cycle, a proliferating pool of cells might beinvolved in disease progression. As a matter of fact,increased clonal expansion of leukemic B-CLL cells inprogressive patients might be due to proliferation inexcess of apoptosis. A central component of thisapoptotic machinery is a proteolytic system thatinvolves a family of proteases called caspases. Thesecaspases can be divided into two groups: largeprodomain containing upstream initiators (caspase-8, caspase-9), which may initiate the proteolytic cascade,and small prodomain containing downstreameffectors (caspase-2, caspase-3, caspase-6), which inturn can amplify the signal by cleaving initiator-caspasesand kill the cell by cleaving key intracellulartargets. In order to define the prognostic impact ofthese apoptotic enzymes on the clinical outcome ofB-CLL, we investigated 178 patients, median age 65years, 88 males and 90 females. Activated caspases2, 3, 6, 8 and 9 were determined on cellular cytosolicextracts through a spectrophotometric detectionof the chromophore p-nitroanilide (pNA) after cleavagefrom the labeled substrates (VDVAD for caspase-2, DEVD for caspase-3, VEID for caspase-6, IETD forcaspase-8 and LEHD for caspase-9). The pNA lightemission was quantified using a microplate reader at400-405 nm. Caspases activities were evaluated bythe absorbance ratio samples/background readings(O. D. ). A mean value of all caspases activities (2, 3,6, 8 and 9) was calculated for each B-CLL patient. Thethreshold of positivity was set at > 0.082 O. D. medianvalue. With regard to patients characteristics, 53had low Rai stage, 120 intermediate stage and 5 highstage. No significant correlations were foundbetween modified Rai stages or β2-microglobulin andcaspases activity levels. On the other hand, there wasa very significant association between high caspasesactivity levels and lymphocyte doubling time (LDT)