494 / THIRD INTERNATIONAL WORKSHOP ON CYTOKINES

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CTL RAISEDAGAINSTA GENE MODIFIED NON-IMMUNOGENIC MCA SARCOMA ARE EFFECTIVE AGAINST THE WILD-TYPE TUMOR. Np Restifo’. PJ Su’ess. S Karo. A Farber. AL Asher. JJ Mule. a nd SA Rosenber?. Snigery Branch, NCI, NIH, Bethesda, MD 20892, USA.

We have previously demonstrated that the non-immunogenic

EVIDENCE OF A DISTINCT LGL ACTIVATION PATHWAY VIA IL-2 VERSUS ANTI- CD2 ON THE BASIS OF A DIFFERENTIAL INHIBITION BY IL-l: RELATIONSHIP TO TNF

MCA-101 murine sarcoma was unable to present endogenously generated viral antigens to specific CTL (JI, in press). We hypothesized that if this tumor was non-immunogenic because of a presentation defect, correction of this defect would enable us to raise anti-tumor CTL. Transfection of MCA 101 with the gene for mIFN-y resulted in increased MHC class I expression and correction of this tumor’s ability to present endogenously produced model viral antigens (lysis of 81% vs -4% at E:T 1O:l). The tumor antigen presentation defect was also corrected: harvest of CD8+ cells from transduced clones was increased by 6.8-fold and these cells were specifically lytic in a 4 hr “Cr assay (ET; 1O:l TIL and 50~1 LAK.)

Robinet E., Chouaib S. Laboratoire d’lmmunologie Cellulaire, URA 1156 CNRS. lnstitut Gustave Roussy, 94805 Villejuif, FRANCE

Wild-type Gene-modified Wild-type Gene-modified

Lymphokine activated killer (LAK) cells can be induced following stimulation of large granular lymphocytes (LGL) with Interleukind (IL-2). We have previously shown that this induction can be negatively regulated by Intedeukin4 (IL-4). In this study, we demonstrate that the stimulation of LGL through the CD2 pathway (9.1 +9.6 mAbs) can also induce the differentiation of LGL into lAK effecters and Tumor Necrosis Factor-alpha (TNF) secretion, suggesting that this factor may play an autocrine role during the differentiation process. While anti-IL-2 mAbs had no effect on the anti-CD2-induced LAK generation, anti-TNF mAbs exhibited a significant inhibition of LAK activity. Our data indicate that, in contrast to the IL- 2 induced LAK generation, the anti-CD2 triggering of LAK differentiation was not inhibited by IL-4. Interestingly, IL-4 was found to enhance the LAK activity as well as LGL proliferation following activation with anti-CD2 mAbs by a mechanism involving at least in part an increased TNF and Interferon-gamma production. These data further demonstrate the key role of TNF in non-MHC restricted killers generation and show that IL-4 serves as a descriminatory factor between 2 distinct pathways involved in the activation of non-MHC restricted cytotoxicity.

TIL 101 ldi+exoe~ 101.22H 102 lOi;exoev 102.228

8 58 66 10 15 22 LAK 74 80 18 18 15 62 Thus, TIL from a gene-modified non-immunogenic sarcoma 101 were lytic specifically against both the gene-modified 101.22H and wild-type 101 pre-treated with exogenous IFN--/. Modification of non-immunogenic tumors with IFN--/ may be useful strategy for developing anti-tumor CTL against tumor histologies not previously susceptible to immunotherapy.

266

IL-6: ANl'I!RMXGCWFHEI'IVITIES INVITROAND INVIVD M. I&ml, Y. Co#d.f, L. CQen, L.M. ii.Nwidr, N.

-, J. C&b&h, Haran- S.,Slavh+, T. Fhyun+.

269

SOLUBLE TUMOR NECROSIS FACTOR (TNF) RECEPTORS IN HUMAN

INFLAMMATORY SYNOVIAL FLUIDS (SF). P. Roux-Lombard and J.-M. Dayer. Div. of Immunology and Allergy. University Hospital. 1211 Geneva 4. Switzerland.

Instihteofz+derce, -t 76100, schml,Jenlsslenl, Isael.

Although TNFa has severaJ activities relevant to the pathogen&s of arthrllis, the levels of this cytokine in SF wefe variable and difficuit to correlate with dllaase

-id l-hman rIL-6 inhibits in vitro culcny fonmtim in activity. This may be due to the presence of soluble fragments originating from severaLlinesofhman&eastandovariancarcinoMs.T47D BreastCal-mveahdantII.r6 recqtmshteticllof

distinct TNF receptors (sTNFR I and sTNFR II ) which bind to TNF and inhibit its

soluble IL-6 Rece&rx (fmnurine)furtherenhaKEs~ biological activities. In wder to test this hypothesis, we measured TNFa, sTNFR I inhibitim by II,%, -plasMcytoMgrowth~ - and II in 10 SF, using specific moncclonal antibodies (Hoffmann-La Roche). Both stinnilated. T47D cells -te an activity that antagcnizes IG6 gmmth inhibiticmon T47TJ and0nM.l mydoleukemiccells. sTNFR were pfesent in all SF tested and their concentrations (mean f SD) were

Tixmechanisnby~chIL-6causesgrowth -ted higher in SF from patients with rheumatoid arthritis (RA) as compared to patients diff-tiatim of Ml leukemia has ken studid. IL-6 sctivates LW-w factors (e.g. IRF) and expressicm of a

wkh ankyicsing spordylnis (AS) or osteoarthrii (OA)

mkerofgmes. IL-6 affects factmsbirdirgtothe Patients s7-fdm 1 em siwm II (timi) TNFa (w/ml)

Interf- Respcnsive enharvzr of 2'-5'AS ad symrgizes with RA n=4 23.9 f 15.2 8.6+ 5.3 30.2 f 16.8 endogenous to activate 2'-5'AS and H-2 t?qressim. ?&ivatim of 2'-5'AS ad EN+-birdiq pttems to enhancers

AS n=2 13.4 f 2.0 6.1 f 2.3 Cl5

asealteredinanoPI-differentia~IG6~istantM1clone. OA n=4 7.3 f 2.6 2.3i 0.7 2O.Of3.3 Invivo, rIL-6 inhibits thedwelopmtof Radiation-induced sTNFR I and II were pesent in significant amounts in SF and may therefore AMLinSJL/Jmi~~ardgrcwthof transplantedAML. Inhibitim of several nWastatic solid tumrs byrIG6is similarly antagonize TNFa effect. The ratio between a cytddne and its spechic inhibitors

cdxwx&.B0thdireztandimrnsx-n&iatedmechanisnsaremost may be critical for the biological outcome mediated by this cytokine and may be

267

IN WV0 ACTIVATION OF EOSINOPHIL GRANULOCYTES BY SUBCUTANEOUS (6.c.) INJECTION OF RECOMBINANT INTERLEUKIN 2 flL21 IN PATIENTS WITH SMALL CELL LUNG CANCER ISCLCI. Rivoltini L., Arienti F., Spinazze’ S., Viviani S., Santoro A., Parmiani G. lstituto Nazionale Tumori, 20133 Milan (Italy).

Seven patients with SCLC treated with S.C. injections of IL2 (9 x lo6 Ill/m*/12 h for 2 days, followed by 3 x 10e IU/m*/12 h for 17 days) developed marked aosinophilia (range: 1550-16500/mmsl during immunotherapy (IT), without significant increase of LAK or NK activity in peripheral blood lymphocytes. Thus, phenotype and functional activity of eosinophil granulocytes IEo) were analyzed to evaluate their potential as anti-tumor effecters. A marked decrease of cell density, as assessed by distribution on Metrizamide gradient, was found to correlate with Eo activation in patients undergoing IL2 treatment. Eo showed an increased expression of FcRll ICD321, HLA-DR. C3R (CDllb) and CR1 fCD351. Cytotoxic activity of Eo against allogeneic tumor cells, as assessed by spontaneous and MAbdependent cellular cytotoxicity (ADCCI. was markedly increased during IT. ADCC was significantly blocked by anti-CD32 MAb. Eo from patients under treatment showed an increased in vitro viability and a higher ADCC after in vitro culture with TNF, GM-CSF and LAK rupernatants. Sera from treated patients f31d weak1 improved the viability and ADCC of EO from healthy donors. Eo obtained before IL2 administration or from healthy donors did not show any antitumor activity. Moreover, patients with progressive disease lacked Eo activation. These data suggest that L.C. injection of IL2 activates Eo, which might be involved in the anti-tumor response. (Partially supported by EuroCetus, Amsterdam).

predictive for the evolution of the disease.

270

EFFECT OF PROLONGED 1~2 SUBCUTANEOUS (s.c.) ADMINISTRATION ON THE CIRCADIAN RHYTHM OF CORTISOL AND B ENDORPHIN (END) IN ADVANCED SMALL CELL LUNG CANCER (SCLC). P.M.

l . . VP. F. Rwe(ll.. /sfifufo Nazianale Tumori. Milan, Italy; l Osp?dale S. Gerarda, Mc+ua. Italy. From 9/% to 3/91,7 pts with advanced SCLC in partial remission alter induction chemotherapy were treated witi ,112 9 million IU/sqm twice daily for two days, followed by 3 million IU/sqm lwice dailv lor 17 davs (5dav treabnent cer week). This was a treatment cyde and was repeated for 3 . . times with 3 week rests. To evaluate the circadian rhythm of c&d and fi e&&in, seturn samoles were collected at 6 AM. 4 PM and 12 PM before and at 1 week intervals until the end of -~- ~~~ ,112 cycle. Two umsecutive rib cycles were evaluated. Serum hormonal levels were measured with the RIA method, and data are reported as mea&E. Before ths start ol ,112 thsrapy, a physiological circadian rhythm of wtisol was present in all 7 patient&while END Qthm was ancmalws in l/7 patients. Cortisoi mean levels seen at 4 PM on the first week #are significantly higher than those seen in basal conditions (246i65 vs 94i42 @ml; P&005). Cortisd peak after 112 administratim progressMy deceased wilh time, and its mean levels observed on Ihe 2nd. 3rd and on the 4th week of therapy, even thwgh still greater, were not signifkantly higher than Ihe baseline ones. Finally, coriisd peak observed on the lirst we& ol the second ,112 cycls was not greater than Hal achievsd during the first cne. An inlxBase in END levels at 4 PM greatet than 100% in respect to the values seen at tie same time in basal conditions was observed in 3/7 patients ~1 the first week of treatment. U-@n END progressively decreased during tha subsequent weeks. END mean levels observed at 4 P.M. alter the first week of therapy were higher than Ihe baseline ones (64&3 vs 3&16 pglml), but this diffefence was not statistically significant. These results, by showing that cortisd rise in response tc ,112 progressively decreases with time, would wggest that the ,112 stimulation of cortisol release does not negativdy influence host immune response. On the other hand, END response to rlL2 is not costant in all pts and this could suggest that different mechanisms are involved in regulating cortisd and END rslsase in response to ,112.