Patent Abstract:
the present invention is related to hybrids and hybridomas consisting of a fused tumor cell and a dendritic cell , which is capable of inducing an anti - tumor response in vivo when administered to a subject in need of anti - tumor treatment . a method of activating autologous immune cells in vitro is disclosed , wherein the method comprises co - cultivating a dendritic cell / tumor cell hybridoma or a plurality of dendritic cell / tumor cell hybrids with immune cells from a mammalian subject .

Detailed Description:
the present invention provides dlc or dc / tumor cell hybrids and hybridomas for activating anti - tumor responses . although the specific procedures and methods described herein are first exemplified using a dba / 2 mouse mastocytoma cell line and dlcs or dcs isolated from syngeneic spleen or from bone marrow progenitors , they are merely illustrative for the practice of the invention . analogous procedures and techniques are applicable for the treatment of human subjects , as thereafter exemplified using a human osteosarcoma cell line and blood - derived dlcs or dcs . therefore , dlc or dc / tumor cell hybrids and hybridomas could be used to immunize human patients against their cancer . procedures applicable to the treatment of a human subject would involve the following steps : a sample is provided of the tumor against which an immune response is needed . such a sample can be obtained when the primary tumor and / or its metastases are removed by surgery , as practised for example for cancers of the breast , prostate , colon , and skin . when the treatment of the cancer involves chemotherapy and / or radiotherapy rather than surgery , as practised for example for small cell lung cancer , lymphomas and leukemias , a sample of the tumor can be obtained from a metastatic site , either before treatment or after relapse . examples of easily - accessible tumor sampling sites are the peripheral blood , bone marrow , peritoneal and pleural effusions , lymph nodes and skin . tumor cells can be separated from blood or bone marrow samples , for instance , by a combination of physical , enzymatic and immunological methods . contaminating red blood cells can be removed by osmotic lysis . tumor cells can be concentrated by density centrifugation . tumor cells can be separated from other cells by binding antigen on the tumor cell surface to antibody - coupled magnetic beads , which are then separated from the biological fluid by means of magnets . in negative cell selection , which may be performed prior to positive cell selection , antibodies bind to antigens that are expressed on contaminating cells , and used to deplete the biological fluids of non - tumor cells . in positive cell selection , antibodies bind to tumor - associated antigens , and this binding is used to separate tumor cells from the biological fluids . when tumor cells are separated by means of antibody - coupled magnetic beads , cells can be released from the beads by digestion of the antigen / antibody binding sites with chymopapain or by other means . the resulting separated tumor cells can re - express the tumor - associated antigen after a short time in culture . the tumor cells are expected to contribute genes encoding known and unknown tumor - associated antigens to the hybridoma of the invention . tumor cells can also be separated from solid tissue samples , using a combination of physical , enzymatic and immunological methods . macroscopic peri - tumoral stromal tissue can be removed by dissection prior to reduction of the tumor to a cell suspension . density centrifugations and antibody - mediated separations can then be performed on the cell suspension as described above . the purified tumor cells are then prepared for cell fusion . three types of tumor partners can be prepared : ( i ) primary cultured tumor cells , ( ii ) immortal tumor cells , and ( iii ) drug - sensitive immortal tumor cells . primary cultured tumor cells are purified tumor cells which have been cultured for a limited period of time in the presence of appropriate growth factors . immortal tumor cells are permanent cell lines derived from these primary cultured tumor cells ; such permanent cell lines can be obtained , for instance , after culturing the primary tumor cells for longer periods of time in the presence of appropriate growth factors , or by transducing the primary tumor cells with immortalizing genes . finally , drug - sensitive immortal tumor cells are permanent cell lines derived from spontaneous mutants of immortal tumor cells ; these mutants are selected by culturing the immortal tumor cells in the presence of an appropriate drug . these drug - sensitive immortal tumor cells die when they are exposed to the drug to which they are sensitive . for example , 6 - thioguanine was used to select the murine p815 * mastocytoma cell line described in example 1 , and 5 - bromo - 2 ′- deoxyuridine was used to select the human 143b osteoasarcoma cell line described in example 7 . both cell lines die when cultured in hat - containing medium , as described in examples 3 and 9 . as an alternative , a pre - established immortal human tumor cell line can be used , provided that at least one of the tumor - associated antigens from the patient &# 39 ; s tumor cells are matched to these pre - established immortal tumor cells . a sample is provided with a source of dlcs or dcs . such samples containing these cells or their precursors include for example peripheral blood , cord blood , bone marrow , lymph or accessible lymph nodes ; they may be taken from the patient or from a healthy , hla - compatible donor . from there , two alternatives are available . functionally - competent dlcs or dcs can be purified directly from these samples , using various methods described in the literature . alternatively , functionally - competent dlcs or dcs can be purified after in vitro differentiation of the precursors contained in these samples , which can be done by culturing the latter in the presence of cytokines , as described hereunder . the dlcs or dcs are prepared for cell fusion , in one of the 4 following ways : ( 1 °) primary dlcs or dcs purified directly from blood , lymph or other tissues are maintained in culture for no longer than 24 hours , as described for mouse spleen dlcs in example 2 . ( 2 °) primary cultured dlcs or dcs differentiated from blood , bone marrow or other tissues are cultured for at least 7 days in the presence of cytokines , as described for human blood dlcs in example 8 or as published by sallusto and lanzavecchia ( j . exp . med . 179 , pp . 1109 - 111 ( 1994 )); romani et al . ( j . exp . med . 180 , pp . 83 - 93 ( 1994 )); mackensen et al . ( blood 86 , pp . 2699 - 2707 ( 1995 )). ( 3 °) immortal dlcs or dcs can be derived from primary - cultured dlcs or dcs , for example by adapting the method described by paglia et al . ( j . exp . med . 178 , pp . 1893 - 1901 ( 1993 )). these authors immortalized neonatal mouse spleen dlcs or dcs by using a recombinant retrovirus . ( 4 °) hat - sensitive variants of these dlc or dc lines can thereafter be derived by standard culture techniques , to yield drug - sensitive immortal dlcs or dcs . a tumor cell partner is then fused with a dlc partner . from there , two alternatives are available , namely to separate or not to separate the fused cells by metabolic selection . after fusion , the treated cells include a plurality of dlc / tumor cell hybrids , as well as unfused tumor cells and unfused dlcs . if no selection is applied , fused cells as well as unfused cells are used for inducing an anti - tumor immunity in vivo and / or in vitro . if a metabolic selection is applied , for example by plating the treated cells in hat - medium , only the immortal , hat - resistant hybrid cells survive ( examples 3 and 9 ) and permanent cell lines hereafter termed dlc or dc / tumor cell hybridomas are developed from them . the dlc or dc / tumor cell hybridomas with therapeutic potential are then selected from all growing hybridomas . their therapeutic potential is linked to the retention of pertinent dlc or dc characteristics and of pertinent tumor cell characteristics . pertinent dlc or dc characteristics include dlc or dc morphology , dlc or dc surface markers , dlc or dc genetic markers and the capacity to activate immune cells in vitro . at least one of these dlc or dc characteristics may suffice to qualify hybridomas made of ( drug - sensitive ) immortal tumor cells and primary cultured dlcs or dcs , since these hybridomas necessarily inherited immortality from the tumor parent . ( 1 °) the selection may be based on the morphologic dlc or dc appearance of the hybridoma by scanning electron microscopy ( sem ), as shown in example 4a and fig1 . such an analysis can be performed on a minute sample of cells at a very early stage of hybridoma development , allowing the culture efforts to be focused on the dendritic - like or dendritic hybridomas . ( 2 °) in the absence of morphological dlc or dc characteristics , as in example 10a , the expression of dlc or dc surface markers may be used to select hybridomas with therapeutic potential . if such dlc or dc surface markers , including namely t - cell activating molecules , are not expressed on resting hybridomas , they may nevertheless be induced by treatment with cytokines or other activating agents , as described in example 10b . ( 3 °) genetic dlc or dc markers are further used to confirm or to exclude the contribution of a t - cell , b - cell or other cell type to the hybridoma , as in examples 4c and 10c . hla - dr gene typing can also be used to identify blood donor genes when the tumor cell and the dlc are from distinct individuals , as in example 10c . in dlc or dc / tumor cell hybridomas involving patient &# 39 ; s related pre - established immortal tumor cells , it is necessary to select dendritic - like hybridomas that express in addition at least one of the patient &# 39 ; s matched tumor - associated antigens . standard immunocytochemistry can be performed on small samples of the hybridomas to identify such tumor - associated antigens as her2 / neu for breast cancer and carcinoembryonic antigen ( cea ) for colon cancer . the hybridomas identified as potentially useful are amplified in culture for complete phenotypic characterization ( chromosomes , genetic markers , cell surface markers and sub - cellular morphology ) and for clinical use . the various embodiments of the invention are briefly described as follows : primary cultured patient &# 39 ; s tumor cells are fused with primary cultured dlcs or dcs purified from blood , lymph or other tissue ( a ), or with primary cultured dlcs or dcs differentiated from precursors derived from blood , bone marrow or other tissue ( b ), or with immortal dlcs or dcs ( c ), to yield a plurality of dlc or dc / tumor cell hybrids that are used without selection . primary cultured patient &# 39 ; s tumor cells are fused with immortal dlcs or dcs ( embodiment d ) or with drug - sensitive immortal dlcs or dcs ( embodiment e ) to yield a plurality of dlc / tumor cell hybridomas ; the latter are mixed in embodiment d with unfused immortal dlc or dc . in these embodiments , hybridomas with both dlc or dc characteristics and tumor cell characteristics may be selected for further use . patient &# 39 ; s immortal tumor cells are fused with primary cultured dlcs or dcs purified from blood , lymph or other tissue ( f ), or with primary cultured dlcs or dcs differentiated from precursors ( g ), to yield a plurality of dlc or dc / tumor cell hybridomas , mixed with unfused immortal tumor cells . in these embodiments , hybridomas with dlc or dc characteristics are selected for further use . patient &# 39 ; s drug - sensitive immortal tumor cells are fused with primary cultured dlcs or dcs purified from blood , lymph or other tissue ( h ), or with primary cultured dlcs or dcs differentiated from precursors ( i ), to yield a plurality of dlc or dc / tumor cell hybridomas . in these embodiments , hybridomas with dlc or dc characteristics are selected for further use . patient &# 39 ; s related , pre - established immortal tumor cells are fused with primary cultured dlcs or dcs purified from blood , lymph or other tissue ( j ), or with primary cultured dlcs or dcs differentiated from precursors ( k ), to yield a plurality of dlc or dc / tumor cell hybridomas , mixed with unfused immortal tumor cells . in these embodiments , hybridomas with dlc or dc characteristics and expressing in addition the patient &# 39 ; s matched tumor - associated antigen ( s ) may be selected for further use . patient &# 39 ; s related , pre - established , drug - sensitive immortal tumor cells are fused with primary cultured dlcs or dcs purified from blood , lymph or other tissue ( l ), or with primary cultured dlcs or dcs differentiated from precursors ( m ), to yield a plurality of dlc or dc / tumor cell hybridomas . in these embodiments , hybridomas with dlc or dc characteristics and expressing in addition the patient &# 39 ; s matched tumor - associated antigen ( s ) may be selected for further use . the selected hybridomas are then used for inducing an anti - tumor immunity , either in vivo or in vitro , thereby contributing to the rejection of the residual tumor in the patient . for the induction of an anti - tumor immune response in vivo , the dlc or dc / tumor cell hybridomas are irradiated or otherwise inactivated , and injected , for example sub - cutaneously , into the patient . the patient is monitored for signs of an anti - tumor immune response and for the clinical evolution of his / her cancer . in a murine model , a single injection of a living dlc / tumor cell hybridoma into syngeneic mice elicited an anti - tumor immune response as shown in examples 5a and 5b . in addition , multiple injections of an irradiated dlc or dc / tumor cell hybridoma had a therapeutic effect on mice preinoculated with a lethal dose of tumor cells , as shown in example 5c . for the induction of an anti - tumor immune response in vitro , the dlc or dc / tumor cell hybridomas are irradiated or otherwise inactivated , and cultured with the immune cells of the patient . the activated immune cells are then re - injected into the patient . the patient is monitored for the presence of an anti - tumor immune response and for the clinical evolution of his / her cancer . the p815 - x2 cell line was derived from the methylcholanthrene - induced mastocytoma p815 of mouse dba / 2 origin ( dunn and potter , 1957 , j . natl . cancer inst . 18 : 587 - 601 . this cell line was obtained by thierry boon , director of the ludwig institute for cancer research , brussels branch , belgium , and recloned by his group ( uyttenhove et al , 1980 , j . exp . med 1562 : 1175 - 1183 ). the subclone p1 was extensively used by t . boon &# 39 ; s group and given to the present inventors in 1980 . a 6 - thioguanine - resistant mutant was derived from p1 , as described by le et al , 1982 , proc . natl . acad . sci . usa 79 : 7857 - 7861 . briefly , p1 cells were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( grand island biological co ., grand island , n . y .) supplemented with 10 % fetal calf serum ( fcs ) ( gibco brl , merelbeke , belgium ), in a 7 % co 2 atmosphere . increasing concentrations of 6 - thioguanine ( sigma , bornem , belgium ), ranging from 1 μg / ml to 30 μg / ml were added to the culture . the final 6 - thioguanine - resistant cells died in hat - medium , i . e . in medium supplemented with 10 − 4 m hypoxanthine , 3 . 8 × 10 − 7 m aminopterin , and 1 . 6 × 10 − 5 m 2 - deoxythymidine ( hat supplement , gibco brl ). several hat - sensitive clones were isolated by limiting dilution from these 6 - thioguanine - resistant cells . a hat - sensitive clone expressing mhc class i antigens was used in the present invention and will hereafter be called p815 *. p815 * cells were cultured at 37 ° c . in a 7 % co 2 atmosphere in tissue culture flasks ( becton dickinson , c a ) containing rpmi 1640 medium ( seromed biochem k g , berlin , germany ) with 10 % fcs ( gibco brl ). one day before use , p815 * cells were diluted with fresh medium in order to be in exponential growth phase at the time of cell fusion . the preparation of splenic dlcs was done according to a multi - step procedure initially described by crowley et al , 1989 , cell . immunol . 118 : 108 - 125 . this procedure was adapted as described by sornasse et al , 1992 , j . exp . med 175 : 15 - 21 . the procedure was started one day before the fusion experiment and yielded 200 , 000 to 500 , 000 dlcs per spleen . briefly , dba / 2 mice were obtained from charles river , sulzfeld , germany , and maintained in specific pathogen - free conditions . animals 8 to 10 weeks old were killed by cervical dislocation ; their spleens were quickly removed and kept in cold rpmi 1640 medium . the spleens were digested with collagenase ( clsiii ; worthington biochemical corp ., freehold , n . j .) and separated into low and high density fractions on a bovine serum albumin gradient ( bovuminar cohn fraction v powder ; armour pharmaceutical co ., tarrytown , n . j .). low - density cells were cultured during 2 hours in rpmi 1640 medium with 10 % fcs , and the non - adherent cells were removed by vigorous pipetting . the latter were further cultured for 1 hour in serum - free rpmi 1640 medium . the non - adherent cells were removed by gentle pipetting and cultured overnight in rpmi 1640 medium with 10 % fcs . the final non - adherent fraction contained at least 95 % dendritic cells , as assessed by morphology and specific staining . the procedure used to fuse hat - sensitive tumor cells with mortal splenic dlcs was adapted from procedures used in our laboratory to generate monoclonal antibodies , as described by franssen et al , protides of the biological fluids , editor h . peeters , pergamon press , oxford , 1982 , pp 645 - 648 . briefly , splenic dlcs and p815 * cells were extensively washed in serum - free rpmi 1640 medium . five million dlcs were mixed with the same number of hat - sensitive p815 * cells in a 15 ml conical tube and centrifuged . two hundred μl of a 50 % solution of polyethylene glycol ( peg 4000 , merck a g , darmstadt , germany ) in rpmi 1640 medium were added dropwise to the cell pellet . the fusion was then stopped by the stepwise addition of rpmi 1640 medium . the cells were washed to remove the peg and resuspended in rpmi 1640 medium with 10 % fcs . after 2 hours incubation at 37 ° c ., the cells were centrifuged , resuspended in rpmi 1640 medium containing hat and 10 % fcs , and plated at 10 4 cells / well in flat - bottomed 96 - well plates ( becton dickinson , c a ). the plates were seeded one day before use with a feeder layer consisting of 5 , 000 irradiated peritoneal cells / well . peritoneal cells were taken from balb / c mice and irradiated at 2 , 000 rads from a cobalt 60 source before plating . the plated fusion was cultured at 37 ° c . in a 7 % co 2 atmosphere . the medium ( rpmi 1640 with 10 % fcs and hat ) was renewed as required by cell growth . in these conditions , unfused dlcs , that are not immortal , died within a few days of culture ; unfused p815 * cells , that are immortal but hat - sensitive , died in the hat - containing - medium , and only hybrid cells , combining the immortality of p815 * cells with the hat - resistance of dlcs survived and developed into growing dlc hybridomas . after 3 - 4 weeks of culture , wells that contained a growing dlc hybridoma could be clearly identified by phase contrast microscopy the content of a positive well was transferred into a larger well ( 24 - well plates , becton dickinson , c a ) previously seeded with irradiated peritoneal cells . eventually , dlc hybridomas were transferred to small tissue culture flasks ( becton dickinson , c a ) and amplified for characterization and storage in liquid nitrogen . the goal of these experiments was to select dlc / tumor cell hybridomas exhibiting at least one of the three following characteristics p815 * tumor cells , fresh splenic dlcs , and dlc / tumor cell hybridomas were analyzed by scanning electron microscopy ( sem ). about one million cells were fixed in 2 - 4 % glutaraldehyde for 24 hours at room temperature and washed in phosphate buffer saline . cell suspensions were then collected on 0 . 2 μm nylon filters , postfixed in 1 % osmium tetroxide followed by 1 % tannic acid mordant and uranyl acetate , with a series of saline washes in between each step . the samples were dehydrated through graded alcohols , then critical point dried from co 2 . after critical point drying , the samples were mounted on aluminium stubs and sputter coated with gold using a bio - rad ps3 coating unit . the cells were examined at 20 kv in a hitachi s520 scanning electron microscope . photographs of the cells are shown in fig1 . in these conditions , p815 * tumor cells appeared as uniform rounded cells , whose surface was spiked with numerous short microvilli ( fig1 a ). in contrast , splenic dlcs appeared as irregular cells , due to the presence of clearly - visible cytoplasmic extensions , resembling pseudopodia and veils . furthermore , the dlc surface was not spiked with numerous microvilli , but displayed instead fewer , larger protrusions . the hybridoma cells were in general much larger than the parent p815 * cells . many of them ( like the one named hyl ) looked very much like the p815 * parent , which was linked to their round regular shape and microvilli - like protrusions ( fig1 c ). in contrast , hybridomas hy41 and hy62 looked much more like the dlc parent , when considering their irregular shape and relatively bare cell surface with some large protrusions , as shown for hy41 in fig1 d . however , a dlc morphology may be assumed not only by dendritic cells of myeloid origin , but also by cells derived from other lineages , including cells of the b - and t - lymphocyte lineages , like follicular dendritic cells and dendritic epidermal t - cells , respectively . in order to determine the cell lineage of the dlc that fused with the p815 * tumor cell , other dlc characteristics were investigated for hybridomas hy41 and hy62 . cell surface molecules were characterized by facs analysis , as described by flamand et al , 1990 , j . immunol 144 : 2875 - 2882 . briefly , the cells were preincubated with 2 . 4g2 , a rat anti - mouse fc - receptor ( fc - r ) monoclonal antibody ( mab ) for 10 min prior to staining with fluorescein - coupled monoclonoal antibody ( fl . mab ). this preincubation was done to prevent the non - specific binding of mab to cellular fc - r . when unlabelled mab were used , they were revealed by incubation with fluoresceinated anti - igg antibodies . the labelled cells were gated for size and side scatter to eliminate dead cells and debris , and analyzed on a facscan ( becton dickinson , c a ). the results are summarized in table 1 . no t - cell activating molecules or other dendritic - cell - associated molecules were expressed by the hy41 and hy62 hybridomas . however , a fraction of the cells of both hybridomas expressed surface cd3e chains of the t - cell receptor ( tcr ), suggesting that they were t - lymphocyte / tumor cell hybridomas . after cloning by limiting dilution , cd3 + and cd3 − subclones were isolated from both hybridomas . fig2 shows that the hy41 and hy62 cd3e + subclones were also labeled by a fl mab specific for the v b8 domain of the tcr , whereas p815 * tumor cells and the cd3e - subclones remained unstained these results showed that the hy41 and hy62 hybridomas expressed an a / b tcr , and hence had incorporated a dendritic - like t - lymphocyte however , neither cd4 or cd8 were expressed by the hybridomas . in order to confirm these cell surface marker studies , genetic marker studies were undertaken . first , southern blot analysis was used to analyse the rearrangement status of the tcr genes in genomic dna from the hy41 hybridoma . the mouse t - cell hybridoma 13 . 26 . 8 - h6 was used as a reference for rearranged tcr genes ( ruberti et al , 1992 , j . exp . med . 175 : 157 - 162 ), and p815 * mastocytoma cells as well as dba / 2 spleen cells were taken as controls for germ line tcr genes . genomic dna was extracted from 2 × 10 7 cultured cells and from spleens , using the genome dna kit ( bio 101 , ca , usa ) according to the manufacturer &# 39 ; s instructions . 10 mg of dna were digested for ± 4 hours with various restriction enzymes , separated on a 1 % agarose gel and transferred to a nylon membrane ( qiabrane nylon plus , qiagen , hilden , germany ) according to standard procedures . the blot was hybridized to a dig - labeled synthetic oligonucleotide of 50 bases targeted to the first exon of the constant region of the mouse tcr b chain and processed for chemiluminescent detection using boehringer mannheim &# 39 ; s dig detection kit . the results showed that the hy41 genome contained a rearranged tcr b chain gene , which is a hallmark of t - cell lineage commitment ( not shown ). next , the polymerase chain reaction ( pcr ) was used to detect rearranged v b8 - cb sequences of the tcr in genomic dna . the upstream primer was targeted to bases 47 - 66 with respect to the atg initiation codon of the mouse v b8 region ( 5 ′- aacacatggaggctgcagtc - 3 ′) and the downstream primer was targeted to bases 141 - 160 of the fisrt exon of the cb region ( 5 ′- gtggacct ccttgccattca - 3 ′). the pcr was carried out essentially according to the instructions of boehringer mannheim &# 39 ; s long range expand pcr system . analysis of the pcr products on a 1 % agarose gel stained with ethidium bromide is shown in fig3 . a fragment with the expected length ( 4 . 5 to 5 kb ) of the rearranged vb8 - cb fragment is clearly seen in dna from the t - cell hybridoma 13 - 26 - 8 - h6 ( lane t ), used as a positive control , as well as in dna from the hy41 and hy62 hybridomas ( lanes 41 and 62 ); this fragment is not amplified in dna from p815 * tumor cells and from spleen cells ( lanes p and s ), used as negative controls . these results confirm that the dlc that fused with a p815 * tumor cell to yield the hy41 and hy62 hybridomas was a t - lymphocyte expressing an a / b tcr receptor , including the vb8 domain . these hybridomas will hereafter be termed t - dlc / tumor cell hybridomas . in conclusion , the hy41 and hy62 t - dlc / tumor cell hybridomas were selected for further studies because of their dlc morphology and t - lymphocyte lineage . in both hybridomas , the t - lymphocyte fusion partner was a rare and undetectable contaminant of the splenic dlc preparation . in view of the complex genetic regulations controling cd4 and cd8 expression in somatic cell hybrids ( wilkinson et al , 1991 , j . exp . med . 174 : 269 - 280 ), it is impossible to determine a posteriori if the fusing t - cell was a cd4 + , cd8 + , or cd4 - cd8 -“ double negative ” t - cell . however , whatever the sublineage of t - lymphocyte involved , the next step was to determine the in vivo immunogenicity of these t - dlc / tumor cell hybridomas . the goal of these experiments was to determine if the hybridomas induced an efficient immune rejection in vivo , as measured by the following criteria : ( 2 °) vaccination with the hybridomas against a subsequent inoculation of tumor cells ; ( 3 °) treatment with the hybridomas after prior inoculation of tumor cells . groups of 10 to 12 dba / 2 mice were injected intra - peritoneally with 500 , 000 living cells of the p815 * tumor or of the hy41 hybridoma . injected animals included mice immunosuppressed by sub - lethal irradiation as well as immunocompetent mice . all irradiated animals died from their tumor within four weeks of inoculation , showing that the hy41 and p815 * cell lines were very similar in their tumorigenicity ( fig4 ). in contrast , 9 / 12 ( 75 %) immunocompetent animals injected with the hy41 hybridoma survived two months after inoculation , when only 2 / 10 ( 20 %) mice had survived the parental tumor injection . this experiment showed that the hy41 hybridoma was as tumorigenic as the parent tumor in irradiated mice , but more immunogenic than p815 * in immunocompetent mice . similar results were obtained with hybridoma hy62 ( not shown ). the 9 surviving hy41 - treated mice , as well as 9 untreated animals , were challenged intra - peritoneally with 500 , 000 p815 * cells . all ( 9 / 9 ) untreated mice died from their tumor within six weeks of inoculation , showing that the tumor cell injection was lethal for unimmunized animals . by contrast , 7 / 9 hy41 - treated animals were still alive 6 weeks after tumor challenge , and 4 / 9 of them survived for at least three months ( fig5 ). these results strongly suggested that prior treatment of syngeneic mice with living hy41 dc hybridoma cells induced a memory immune response against the parent p815 * cell line , conferring tumor resistance to 44 % of the treated animals . a similar tumor - resistance could be induced by the injection of living hy62 hybridoma cells ( not shown ). the spleens of the 4 p815 *- resistant mice were tested in vitro for the presence of anti - p815 * cytotoxic t - cells , as described by moser et al , 1987 , j . immunol 138 : 1355 - 1362 . briefly , spleen cell suspensions were stimulated in vitro during 5 days with the irradiated p815 * cells , in order to induce a measurable memory response . they were then used as effector cells on chromium - loaded p815 * and l1210 target cells . the latter have the same mhc class i haplotype ( h - 2d ) as p815 cells . at several effector / target ratios , the spleen cells of the untreated animals completely failed to lyse the p815 * and the l1210 target cells ( fig6 a and 6b ). in contrast , the spleen cells from the 4 p815 *- resistant mice lysed efficiently and specifically the p815 * targets , without showing any significant activity on the l1210 targets . these results showed that the hy41 - treated , p815 *- resistant animals were able to mount a strong and tumor - specific cytolytic response upon in vitro restimulation . in this experiment , 40 dba / 2 mice received an ip injection of 2 × 10 5 p815 * tumor cells . seven days later , the mice were divided into 4 groups of 10 animals ; the first group was left untreated while the 3 other groups were treated by 4 weekly ip injections of 2 × 10 6 irradiated ( 15 , 000 f ) p815 *, hy41 or hy62 cells . the data are presented in fig7 . untreated animals all died within 7 weeks of tumor inoculation , and 20 % of the mice treated with irradiated p815 * tumor cells survived , confirming the weak immunogenicity of the p815 tumor cell line . in contrast , 60 % and 40 % of the mice treated with irradiated hy41 and hy62 hybridoma cells , respectively , survived the prior injection of a lethal dose of p815 * cells . these data showed that hybridoma hy41 , and to a lesser extent hybridoma hy62 , could induce the immune rejection of an established tumor . however , the mechanism leading to such an efficient in vivo immune rejection remained unclear . one possibility that was explored concerned the secretion of immunomodulating cytokines . the goal of these experiments was to determine whether the hy41 and hy62 hybridomas synthesized some cytokines that could account , at least in part , for their in vivo immunogenicity . total rna was prepared from activated spleen cells , from p815 * tumor cells and from the hy41 and hy62 hybridomas according to standard procedures . the reverse - transcription polymerase chain reaction ( rt - pcr ) and cytokine - specific primers were used to amplify il - 2 , il - 4 , il - 10 and interferon γ ( ifn - γ ) mrna sequences , as described by de wit et al , j . immunology , 1993 , 150 : 361 - 366 . the primers used to amplify il - 12 p40 sequences were 5 ′- ttcaacatcaagagcag tagc - 3 ′ and 5 ′- ggagaagtaggaatggggagt - 3 ′. analysis of the rt - pcr products on ethidium bromide - stained agarose gels showed that p815 * tumor cells constitutively expressed il - 4 mrna and that the hy41 and hy62 hybridomas constitutively expressed il - 2 and il - 4 mrnas , but not il - 10 , il - 12 , and ifng mrnas . these cytokine mrnas were nevertheless detected in activated spleen cells , used as a positive control . in conclusion , these data showed that the hy41 and hy62 t - dlc / tumor cell hybridomas constitutively expressed il - 4 like the parent p815 * tumor cell , and il - 2 , like the parent t - lymphocyte . these cytokines , if secreted in vivo , may at least partially contribute to the immunogenicity of the hybridomas . the human 143b thymidine kinase negative osteosarcoma cell line ( hereafter termed 143b ) is a hat - sensitive cell line that was purchased from the atcc ( crl n ° 8303 ). the cells were cultured in dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % fcs , 2 % penicillin / streptomycin , 1 % sodium pyruvate ( all from gibco brl , merelbeke , belgium ) and 0 . 015 mg / ml of 5 - bromo - 2 ′- deoxyuridine ( sigma chemical co , st louis , mo .). one day before fusion , the cells were diluted with fresh medium in order to be in exponential growth phase . dendritic cells were differentiated in vitro from adherent blood precursors , using an adaptation of the technique described by romani et al , 1994 , j . exp . med . 180 : 83 - 93 . briefly , peripheral blood mononuclear cells ( pbmc ) were isolated from the buffy coat of a healthy donor by density gradient centrifugation on lymphoprep ( gibco brl ). adherent cells were prepared by plating 10 7 pbmc on 6 - well tissue culture plates in 3 ml rpmi supplemented with 200 mm l - glutamine , 50 mm mercaptoethanol and 10 % fcs . after 2 hours incubation at 37 ° c ., the non - adherent cells were discarded by a very gentle rinse , and the adherent cells were further cultured in the above - described medium supplemented with gm - csf ( leucomax , 800 u / ml ) and il - 4 ( genzyme , 500 u / ml ), at 37 ° c . in a humidified atmosphere with 5 % co 2 . after 7 days of culture , dlcs were recovered and characterized by cell scatter and cell surface marker analysis . the dlcs used for fusion contained 50 % of monocytic - like cells , expressing cd14 but not cd1a or cd1c , as well as 38 % of t - lymphocytes , 4 % of nk cells and 8 % of b lymphocytes . the procedure used to fuse hat - sensitive tumor cells with dlcs was adapted from procedures used to generate monoclonal antibodies ( current protocols in immunology , chapter 2 . 5 . 4 ). the 143b tumor cells and the dlcs were extensively washed in serum - free medium ( rpmi 1640 ); 2 × 10 6 dlcs , were mixed with 1 × 10 6 143b osteosarcoma cells and centrifuged . the pellet was resuspended in 500 μl of a 50 % solution of polyethylene glycol ( peg 4000 , gibco ) in dulbecco &# 39 ; s phosphate buffered saline without ca ++ , mg ++ ( ref . 14030035 ). after 1 minute , the peg was progressively diluted by the slow and progressive addition of serum - free medium . the cells were washed free of peg and resuspended in rpmi 1640 with 10 % fcs . they were eventually plated at 2 × 10 4 cells / well in flat - bottomed 96 - well plates ( falcon , becton dickinson ) and cultured in a 5 % co 2 atmosphere at 37 ° c . hat medium was added to the wells 24 hours after fusion and renewed every two days . in these conditions , unfused dlcs died within 2 - 3 weeks of culture , unfused 143b osteosarcoma cells died in hat - medium and only hybrid cells combining the immortality of the tumor cell with the hat - resistance of a dlc survived and developed into growing cell lines . after 3 - 4 weeks of culture , wells containing growing cell lines were clearly identified by phase contrast microscopy . their contents were transferred into larger wells and eventually into culture flasks for amplification . culture stocks were frozen in liquid nitrogen before analysis . the goal of these experiments was to identify human dlc / tumor cell hybridomas presenting at least one of the three following characteristics : the 143b osteosarcoma cells and a series of hybridoma cells were analyzed by sem , as described in example 4 . comparison of the parent cells and hybridoma cells showed that none of the hybridomas analysed , including f3bg10 cells , displayed morphologic dendritic - like features . in the absence of such features , other dendritic - like features were analyzed , namely the presence of dlc surface markers . cell surface markers were analyzed as described in example 4 . results are summarized in table 2 . none of the tested hybridomas , including f3bg10 cells , expressed the t - cell activating molecules hla - dr , b7 . 1 , and b7 . 2 . however , they expressed hla class i , icam - 1 ( cd54 ) and lfa - 3 ( cd58 ), which were also present on the 143b tumor cells . they failed to express typical dendritic - cell markers like cd1a and cd1c , as well as markers specific for t - cells ( cd3 ), b - cells ( cd19 ), nk cells ( cd56 ) and monocytes ( cd14 ). since the hybridomas tested failed to express constitutively t - cell activating molecules , they were stimulated with a variety of cytokines in order to induce such expression . it was found that 40 % of the f3bg10 hybridoma cells were induced to express varying amounts of surface hla - dr after a 24 hour incubation with interferon γ . after cloning by limiting dilution , subclones were tested for their capacity to express induced hla - dr . fig8 shows that at least 90 % of h12 cells clearly expressed induced hla - dr , which greatly increases their immunogenic potential . the goal of this first experiment was to determine whether the f3bg10 hybridoma had been generated by the fusion of a dlc with the 143b tumor cell , and to exclude that it was a revertant 143b tumor cell clone , that had become resistant to hat - medium by mutation . this was done by typing the hla - dr genes of the blood donor , of the 143b tumor cell and of the f3bg10 hybridoma . genomic dna was prepared according to standard procedures from 143b tumor cells , from the pbmc of the blood donor and from f3bg10 hybridoma cells . these dnas were submitted to a non - isotypic hla - dr b oligotyping method , described for the typing of dr b 1 , 3 , 4 , 5 alleles by buyse et al . 1993 , tissue antigens 41 : 1 - 4 . the polymorphic second exon of the corresponding genes was amplified by pcr , and biotinylated nucleotides were incorporated into the amplifying fragments during this procedure . the pcr products were hybridized with a combination of 31 sequence - specific oligonucleotide probes , immobilized in parallel lines on membrane strips . after a stringent wash , streptavidin - labelled alkaline phosphatase was added to mark the biotinylated dna fragments . the addition of the bcip / nbt chromogen resulted in a colored precipitate . all reagents were part of the innolipa drb key kit purchased from innogenetics ( zwijndrecht , belgium ). the f3bg10 lane showed a mixture of bands corresponding to alleles present in the 143b osteosarcoma cells and in the pbmc of the blood donor , confirming that f3bg10 hybridoma was a dlc / tumor cell hybridoma . the goal of the second experiment was to investigate whether it was a t - lymphocyte or a b - lymphocyte that fused with a 143b tumor cell to yield the f3bg10 hybridoma . genomic dna was tested for the presence of rearranged t - cell receptor ( tcr ) genes or b - cell receptor ( bcr ) genes by southern blot analysis with tcr - specific or bcr - specific probes . standard procedures were used . briefly , samples of 10 mg of dna were submitted to overnight digestion at 37 ° c . with different restricition enzymes . hind3 , xba1 and hind3 + xba1 were used for the tcr rearrangements and ecor1 , hind3 and hind3 + bamh1 were used for bcr rearrangements . the restriction fragments were separated by electrophoresis on a 1 % agarose gel , transferred to nitrocellulose , baked and hybridized with probes specific for either the b chain gene of the tcr , or for a segment of the j gene of the ig heavy chain of b lymphocytes . the results clearly showed that there were only germ line tcr genes and germ line bcr genes in the genomic dna of the f3bg10 hybridoma . these data excluded that the dlc / tumor cell hybridoma f3bg10 was produced by the fusion of the tumor cell with a t - lymphocyte or a b - lymphocyte . the dlc fusion partner could have been a monocyte , a dendritic cell , an intermediate cell between these two cells , a natural killer cell or another unidentified non - b cell . because the pattern of cytokine secretion could provide indications on the cell lineage of the fusion partner , we investigated cytokine secretion by f3bg10 cells . in vitro analysis of cytokine secretion by human dlc / tumor cell hybridoma the culture supernatants of the f3bg10 hybridoma cells and of the 143b tumor cells were assayed by elisa for the presence of various cytokines , before and after 36 hours of culture in the presence of various stimuli including interferon γ , tnfα , gm - csf and combinations of these . the results showed that the 143b osteosarcoma cells and the f3bg10 hybridoma cells secreted similar levels of il - 6 and il - 8 , that could be increased for both cytokines by stimulation with the above - mentioned cytokines . in addition , the f3bg10 cells but not the tumor cells secreted significant levels of gm - csf , that could be increased by stimulation . neither the tumor cells or the hybridoma cells secreted detectable levels of il - 1β , il - 10 , il - 12 and tnfα . these results showed that the f3bg10 hybridoma secreted il - 6 and il - 8 like the parent tumor cell , and gm - csf like the parent dlc . since it was excluded that the latter was a t - lymphocyte , this result suggested that the fusion partner was a monocyte . female dba / 2 ( h - 2 d ) and cba / j ( h - 2 k ), 6 - 8 week old , were purchased from charles river wiga ( sulzfeld , germany ) and maintained in our own pathogen - free facility . the tumor cell line is the methylcholanthrene - induced mastocytoma p815 of dba / 2 origin , derived from a 6 - thioguanine - resistant mutant , according to a procedure described by lethé et al . ( 1992 ). briefly , p815 cells were cultured in dmem supplemented with 10 % fcs and increasing concentrations of 6 - thioguanine ( sigma , st . louis , mo . ), ranging from 1 to 30 μg / ml . the final 6 - thioguanine - resistant cells died in hat - medium , i . e . in medium supplemented with 10 − 4 m hypoxanthine ( merck , a g , darmstadt , f r g ), 3 , 8 10 − 7 m aminopterin ( icn nutritional chemicals ) and 1 . 6 × 10 − 5 m 2 - deoxythymidine ( merck a g ). l1210 is a lymphocytic leukemia which arose in a dba / 2 female following painting the skin with methylcholanthrene ( available through atcc ). the i - e d restricted , pork - insulin specific t cell hybridoma b8p4 . 1c3 ( 24 ) was obtained from dr delovitch ( j . p . robarts research institute , ontario , canada ). dendritic cells were generated from bone marrow progenitors according to a procedure modified from a protocol of inaba et al . ( 1992 ) and zorina et al . ( 1994 ). briefly , bone marrow was flushed from tibias and femurs and depleted of lymphocytes , granulocytes and class ii positive cells using a cocktail of mabs and sheep anti - rat igg dynabeads m - 450 ( dynal , oslo , norway ). the mabs were anti - cd8 , anti - cd4 , gr - 1 anti - granulocyte , anti - b220 / cd45r , anti - i - a d / i - e d ( pharmingen , san diego , calif ., usa ). cells were plated in 24 - well culture plates ( 2 . 5 × 10 5 cells / ml / well ) in dmem supplemented with 10 % heat inactivated fcs , additives , 200 ng / ml gm - csf and 100 u / ml tnfα , and cultured for 10 days . the cultures were fed every other day by gently swirling the plates , removing 75 % of medium and adding fresh medium containing gm - csf and tnfα . non - adherent cells were collected at 10 days and comprised mainly dendritic cells , as assessed by morphology and specific staining using n418 ( 26 ), anti - class ii , anti - b7 - 1 ( 9 ) and anti - b7 - 2 ( 10 ) mabs . 2 × 10 6 dc were mixed with 2 × 10 6 hat - sensitive p815 cells in a 15 ml conical tube . the cells were washed in rpmi 1640 and pelleted by centrifugation . the fusion was started by adding dropwise , in 90 seconds , 200 μl of a 50 % solution of peg 4000 ( merck ) in rpmi 1640 medium . the fusion was stopped by the stepwise addition of rpmi medium . the cells were centrifuged , resuspended in medium containing 10 % fcs and additives , and incubated for 2 h , at 37 ° c . in 7 % co 2 . the cells were centrifuged , resuspended in selection medium ( rpmi 1640 containing hat , 10 % fcs and additives ), and plated at 10 4 cells / well in flat - bottomed 96 - well plates ( becton dickinson , c a , usa ). the plates were seeded 1 day before use with a feeder layer consisting of 5 , 000 ( irradiated peritoneal cells / well . the plated fusion was cultured at 37 ° c . in a 7 % co 2 atmosphere . the medium was renewed as required by cell growth . the use of lethally irradiated tumor cells as a therapeutic modality should be transferred readily into clinical application . high numbers of dendritic cells can be derived from progenitors in humans ( caux et al . ( 1992 )). the great majority of tumor antigens are either unknown or indeterminate with regard to their immunogenic t - cell epitopes . furthermore , the method and composition of the invention combine several advantages such as the presence of costimulatory molecules , the ability to present antigen through the exogenous ( mhc class ii ) and endogenous ( mhc class i ) pathways independently from known mhc / epitope associations . of note , presentation of multiple antigen derived epitopes may enhance anti - tumor immunity and minimize the emergence of resistant variants . using dc as an adjuvant for antigen delivery has potential advantages over other forms of immunization in that dc may have the unique property to migrate to areas rich in t - lymphocytes and to express a variety of signals that lead to optimal activation of naive and memory cells . cells were analyzed by flow cytometry with a facscan cytometer ( becton dickinson and co , mountain view , calif .). the cells were preincubated with 2 . 4g2 ( a rat anti - mouse fc receptor mab ) for 10 min before staining to prevent antibody binding to fcr , and were incubated with fluoresceinated 14 - 4 - 4 ( murine igg2a anti - i - e d , available through atcc , rockville , md ., usa ), n418 ( hamster anti - mouse cd11c , 26 ), 16a1 ( hamster anti - mouse b7 - 1 , 9 ), gl1 ( rat igg2a anti - mouse b7 - 2 , 10 ), anti - heat stable antigen ( hsa , pharmingen , san diego , calif ., usa ), anti - mouse icam - 1 / cd54 ( pharmingen ). staining with irrelevant isotype - matched antibodies was negative on all cell types . total rna was extracted from p815 and hybrid cells using trizol reagent ( total rna isolation reagent , gibco brl , merelbeke , belgium ). less than 1 μg rna was used to perform a control pcr for actin and a p1a gene specific pcr with the titantm one tube rt - pcr system ( boehringer mannheim , brussels , belgium ). the cdna synthesis was performed following the manufacturer &# 39 ; s instructions . the pcr reactions for actin : 94 ° c . 2 ′ ( 94 ° c . 30 ″, 60 ° c . 30 ″, 72 ° c . 1 ′ 20 ″) 40 cycles , 72 ° c . 10 ′ and for p1a : 94 ° c . 2 ′ ( 94 ° c . 30 ″, 55 ° c . 30 ″, 72 ° c . 30 ″) 35 cycles , 72 ° c . 10 ′ were in a perkin - elmer / cetus dna thermal cycler . primers used were as follows : actin sense primer 5 ′- tgctatccaggctgtgctat - 3 ′, actin antisense primer 5 ′- gatggagttgaaggtagttt - 3 ′, pla sense primer 5 ′- gggaccatggcccacagtggctcaggt - 3 ′ and p1a antisense primer : 5 ′- gggggatccttagacagaggacatgcgcttg - 3 ′, resulting in an amplified fragment of 240 bp . the complete medium used in all experiments was rpmi 1640 ( seromed biochem k g , berlin , germany ) or dmem ( gibco brl , merelbeke , belgium ) supplemented with 10 % fcs , 2 % ultroser hy ( a serum - free medium supplement purchased from gibco brl ) or 1 % heat - inactivated mouse serum , penicillin , streptomycin , non - essential aminoacids , sodium pyruvate , 2 - me , and l - glutamine ( flow icn biomedicals , bucks , uk ). mixed lymphocytes reaction ( mlr ): splenic cd4 + t - cells ( cba / j , h - 2 k ) were purified by depletion of adherent cells by passage over sephadex g10 ( pharmacia bioprocess , uppsala , sweden ) and complement - mediated lysis with a cocktail of anti - b220 and anti - cd8 mabs . 2 × 10 5 cd4 + t - cells were stimulated with increasing numbers of γ - irradiated ( 15 , 000 rads ) allogeneic p815 or hybrid cells , or with γ - irradiated ( 3000 rads ) bone marrow - derived dc . proliferation was assessed by thymidine incorporation during the last 16 h of a 4 day - culture . the supernatants were collected after 48 h of culture , frozen and assayed for il - 2 content using a standard bioassay with an il - 2 sensitive , il - 4 insensitive subclone of the ctl . l line . in some experiments , purified blocking antibodies were added at a final concentration of 5 μg / ml , as indicated in fig1 . tumor specific immune response : resistant mice ( injected with live p815 and irradiated hybrid cells , and further challenged with live p815 cells harvested from ascites ( see fig1 ) were killed 3 months after the last treatment . 6 × 10 6 splenocytes were stimulated with 10 5 irradiated ( 15 000 rads ) p815 in a volume of 2 ml of dmem containing additives and 2 % ultroser hy . after 5 days of culture , the effectors generated were tested for lytic activity in a 3 . 5 - h 51 cr - release assay on p815 . results are expressed as percent specific lysis at various e / t ratios . percent specific lysis of target cells was calculated as follows : 100 ×( experimental release − spontaneous release )/( maximum release − spontaneous release ). each point represents the mean percent specific 51 cr release from three replicate wells . standard errors were consistently & lt ; 5 % of the mean values . 50 μl of supernatants were collected after 24 h of culture , frozen and assayed for il - 2 content . il - 2 production by cells from the peritoneal cavity was tested as follows : the cells were harvested from the same treated mice by extensive washing of the peritoneal cavity with cold dmem , and 6 × 10 4 peritoneal exudate cells were cultured ( in dmem containing 1 % mouse serum and additives ) with various numbers of irradiated p815 cells in round - bottom 96 - well plates . the supernatants were collected after 48 h of culture and assayed for il - 2 content . cultured tumor cells were washed three times with pbs and resuspended in pbs for implantation into mice . dba / 2 mice were injected intraperitoneally with 2 × 10 5 p815 or 2 × 10 4 l1210 tumor cells . some animals received 3 or 7 injections of 2 × 10 6 irradiated p815 tumor cells or hybrid cells , cultured or not with gm - csf , every 5 days starting on day 3 after tumor inoculation . in the experiment depicted in fig1 , panel b , 2 × 10 5 p815 cells were injected intraperitoneally into sublethally irradiated dba / 2 mice ( 800 rads ) and tumor cells harvested from ascites were used to assess tumor resistance in vivo . one hybrid displayed morphologic and phenotypic features of dendritic cells and expressed mrna specific for p815 - associated antigen p1a . 2 × 10 6 hat sensitive p815 cells were fused with the same number of bone marrow - derived dendritic cells , as described in material and methods . 50 clones proliferated in selection medium containing hat , and one clone , hybrid 38 , displayed morphological features of dendritic cells . as shown in fig9 hybrid cells , cultured with gm - csf , expressed cd11c , mhc class ii and costimulatory molecules ( b7 - 1 , b7 - 2 and hsa ). by contrast , p815 mastocytoma cells and hybrid cells cultured in the absence of gm - csf expressed none of these markers . previous publications have shown that the p1a gene is expressed in p815 mastocytoma and encodes a protein that includes a nonapeptide representing a tumor rejection antigen ( p815ab ; brichard et al . ( 1995 ); lethé et al . ( 1995 )). hybrid 38 has been tested for the expression of mrna specific for p1a and showed that hybrid cells , cultured with or without gm - csf , as well as p815 tumor cells express mrna for p1a , whereas dc generated from bone marrow progenitors were negative ( fig1 ). hybrid 38 is a somatic hybrid ( it contains an average of 73 chromosomes ) between a dendritic cell , as suggested by the phenotype and function ( see below ), and a mastocytoma cell , as assessed by expression of mrna specific for p1a . hybrid 38 and bone marrow - generated dc , but not p815 , induced primary responses in vitro . hybrid cells had the capacity to process and present exogenous antigen in the context of class ii mhc . fig1 shows that t - cell hybridoma secreted high levels of il - 2 when cultured with gm - csf treated hybrid cells and insulin protein . no il - 2 was produced in the absence of insulin . furthermore , since dc appear to have the unique property to activate naive t - cells in vitro , the inventors have tested the capacity of hybrid cells , p815 and bone - marrow derived dc to induce primary immune responses in vitro . irradiated , gm - csf - treated hybrid cells and dc from dba / 2 mice ( h - 2 d ) induced proliferation ( fig1 ) and il - 2 secretion ( fig1 ) by purified cd4 + t - cells from cba mice ( h - 2 k ). by contrast , p815 and hybrid cells cultured in the absence of gm - csf did not sensitize allospecific t - lymphocytes in vitro , as assessed by proliferation and il - 2 secretion at background level . thereafter the role of b7 - 1 and b7 - 2 in the induction of primary response was determined . the addition of neutralizing antibodies specific for b7 - 1 and b7 - 2 abrogated t - cell proliferation and il - 2 secretion ( fig1 d ). antibodies to b7 - 2 alone significantly reduced t - cell activation , whereas anti - b7 - 1 or isotype - matched control antibodies had no effect . repeated injections of hybrid cells prevented the growth of pre - established p815 mastocytoma and induced long - term protection . the potential utility of hybrid - based immunization for the therapy of established tumors was tested in mice inoculated with a lethal dose of p815 intraperitoneally 3 days previously . mice bearing growing tumor received 7 - intraperitoneal injections of 2 × 10 6 irradiated ( 15 , 000 rads ) hybrid cells from day 3 to day 33 after tumor inoculation . this therapy resulted in long - term tumor protection in 55 % ( fig1 ) of the animals . the tumors grew progressively and killed the animals in the control groups that included untreated mice , mice treated with irradiated hybrid cells cultured without gm - csf , or animals injected with irradiated p815 cells . the specificity of tumor resistance induced by hybrid cells was demonstrated by the lack of effect of hybrid therapy on the growth of leukemia l1210 , a methylcholanthrene - induced leukemia of dba / 2 mice ( fig1 panel a ). to test whether 7 injections were required to prevent tumor growth , 3 groups of mice were injected with p815 , two of them were subsequently treated with irradiated hybrid cells . the data show that 3 or 7 injections of hybrid cells resulted in similar protection ( 100 h and 90 %, respectively ) to preinjected p815 ( fig1 panel a ). whether hybrid therapy resulted in long - lasting resistance was tested . to avoid the potential helper effect generated by components of the fcs present during culture of hybrid and tumor cells , surviving mice were subsequently injected with p815 cells harvested from irradiated mice inoculated with mastocytoma cells . the data in fig1 ( panel b ) show that treated mice were protected against a second tumor challenge , whereas all control mice died within 23 days after tumor inoculation . the tumor resistance induced by hybrid cells correlates with the development of il - 2 secreting cells and tumor - specific cytotoxic t - lymphocytes . to characterize the anti - tumor immunity induced by hybrid cells , splenocytes and peritoneal exudate cells from resistant mice ( inoculated with p815 , treated with irradiated hybrid cells and challenged with live p815 harvested from ascites , see fig1 b ) were restimulated in culture with irradiated tumor cells . the data in fig1 show that injection of hybrid cells , cultured with gm - csf , promoted the generation of cells displaying cytotoxic activity to p815 ( panel a ), as well as the development of il - 2 secreting cells in the spleen ( panel b ) and in the peritoneal cavity ( panel c ) these immune responses were dependent on the in vitro restimulation with irradiated p815 cells . no such immune response was detected in untreated mice . a cancer therapy based on the elimination of tumor cells in vivo by the immune system offers several advantages which include antigen specificity , lack of toxicity , ubiquity and immunological memory which should ensure long - term resistance . the approach to improve the tumor - specific immune response is based on the two - signal theory which implies that two distinct signals are required for optimal activation of t - lymphocytes ( schwartz ( 1990 , thompson et al . ( 1995 )). the apcs have therefore a dual function and provide the ligands for the t - cell receptor as well as for the cd28 receptor . since most tumor cells do express specific antigens ( recently reviewed by van den eynde and van der bruggen ( 1997 )) but do not provide the second signal , it was hypothesized that a limiting factor in the tumor - specific immunity could be a defective antigen presentation due to the lack of costimulation this hypothesis is strengthened by recent studies from huang et al . ( 1994 ) showing that the priming of an immune response against an mhc class i restricted antigen that is expressed in non - hematopoietic cells , such as a tumor antigen , involves the transfer of that antigen to a host bone marrow - derived cell before its presentation to cd8 + t - cells . ( i ) dc have been loaded with tumor antigens in the form of proteins , peptides or unfractionated acid eluted peptides and ( ii ) tumor cells have been transduced with genes encoding helper factors or costimulatory molecules ( for review , see young and inaba ( 1996 )). in particular , immunization with irradiated p815 transfected with b7 - 1 gene successfully induced ctl activity in 100 % of mice and protected against tumor challenge ( gajewski et al . ( 1996 )). dc pulsed with p815ab alone did not induce t - cell reactivity , whereas the addition of helper peptides led to efficient priming , suggesting that the failure of p815ab to initiate cd8 + cell reactivity may be due to defective recruitment of helper t - cells to the afferent phase of the response ( grohmann et al . ( 1995 ), bianchi et al . ( 1996 )). the present invention shows that somatic hybrid cells formed between tumor cells and dc have unexpectedly the capacity to provide , both antigenic and costimulatory signals to t - cells and to induce specific protection against the established parental tumor . p815 mastocytoma has been shown to express five distinct antigens ( a , b , c , d , e ) recognized by syngeneic cytolytic lymphocytes ( bricahrd et al . ( 1995 )). two of these tumor rejection antigens , p815a and p815b , are encoded by gene p1a and are presented by class i molecule l d ( van den eynde et al . ( 1991 )) both of which are expressed by hybrid 38 . there is evidence that the antigen p815a / b is of critical importance in the rejection of the tumor , as p815 a and / or b are lost by tumor cells that escape tumor rejection in vivo ( lethé et al . ( 1992 ), brichard et al . ( 1995 )), although antigens cde are also involved in tumor resistance . the inventors have discovered that hybrid cells , but not p815 , may express tumor - associated antigens in the context of class ii , thereby leading to activation of cd4 + cells , whereas both cell populations would express p815 - derived peptides in the context of class i mhc hybrid cells and sensitize cd8 + cells . furthermore , hybrid cells , but not the parental tumor , express b7 and hsa molecules , both of which have been shown to provide the costimulatory signal required for optimal activation of t - lymphocytes . liu et al . ( 1997 ) suggest the induction of memory t - cells requires costimulation by either b7 or hsa , while the induction of effector t - cells depends on b7 but not hsa . the characterization of the spontaneous immune response to p815 in a syngeneic host highlights the critical role of b7 .- cd28 interaction in initiating an antitumor response . an immune response to tumors which do not express b7 is dependent on costimulation by b7 - 1 and b7 - 2 expressed by host cells ( yang et al . ( 1997 )) and requires migration to b7 - expressing - sites , such as lymph nodes or spleens . however , this response is insufficient to inhibit subsequent outgrowth of tumor unless the response is further strengthened e . g . by sensitization against b7 + tumor cells . of note , inhibition of t - cell migration into lymph nodes eliminates the immune response to the b7 − , but not to the b7 + p815 implanted in the hind footpads of mice ( yang et al . ( 1997 )). the spontaneous immune response to tumor of non - hematopoietic origin may therefore depend on trans - costimulation , whereas unexpectedly injection of hybrid cells would give rise to higher immune response ( by cis - costimulation ) and allow initiation of the response at the site of the tumor . the effector cells that mediate the elimination of p815 in vivo most probably involve cytotoxic t - lymphocytes , as well as il - 2 and ifn - γ secreting cells . the tumor resistance induced by hybrid cells correlates with the development of cytotoxic t - lymphocytes in spleen ( fig1 ) as well as il - 2 ( fig1 ) and ifn - γ secreting cells in spleen and at the site of the tumor . more recently , the incidence of a high ifn - γ producing phenotype in draining lymph nodes of mice has been shown to correlate with the frequency of rejection of p815 implanted in the hind footpads ( 47 ). although the same report has underlined the role of il - 12 in rejection of p815 in vivo , no expression of mrna coding for il - 12 by hybrid cells has been detected . an efficient immune response may not only prevent tumor growth in vivo , but also limit the onset of antigenic or mhc - loss variants as well as the mechanisms of suppression by the tumor itself . the immunostimulatory properties of hybrid cells are gm - csf - dependent , as hybrid cells cultured without gm - csf do not express mhc class 11 , b7 nor hsa molecules , do not sensitize naive t - cells in vitro and do not induce tumor resistance in vivo . this observation may be related to the maturation process that is the hallmark of cells from the dendritic family . langerhans cells and dendritic cells have a specialization of function over time and undergo phenotypic and functional changes during a phenomenon of maturation that occurs spontaneously in vitro ( inaba et al . ( 1994 )) and may be induced in vivo ( de smedt et al . ( 1996 )). although the factors that induce this process are largely unknown , gm - csf seems to be involved . experiments are under way to transfect the gene coding for gm - csf in hybrid cells and to test their function in vitro - and in vivo . hybrid cell immunization mediates a specific anti - tumor immunity , since no protection was observed against l1210 lymphoma cells , indicating that carry - over of gm - csf is not the factor inducing tumor rejection . there is evidence that the cd28 costimulatory pathway is functional in nk cells and plays an important role in their proliferation and cytokine production ( geldhof et al . ( 1995 )). of note is that hybrid cells , but not p815 cells , are lak - sensitive targets , suggesting that hybrid 38 may induce or enhance nk activity . in addition , nk cells are known to be potent producers of ifn - γ at an early stage of activation , and may direct the development of a tumor - specific th1 and ctl response . the in vivo depletion of nk cells prior to immunization with melanoma cells has been shown to abrogate the capacity of spleen cells to generate cd8 + tumor specific ctl after in vitro restimulation ( kurosawa et al . ( 1995 )) therefore , innate ( nk ) and adaptative ( ctl ) cytotoxic immune responses appear to be crossregulated and injection of b7 + hybrid cells may lead to enhancement of both responses ( kos and engleman ( 1996 )). bone marrow - derived dc have been shown to combine the high t - cell stimulatory properties with the capacity to process and present native antigens ( garrigan et al . ( 1996 )). fusion experiments have been performed using p815 and dendritic cells isolated from spleen . the yield of hybrid clones was very low , as compared to fusions between p815 and bone marrow - derived dc , and none of them displayed phenotypic and functional features of dendritic cells , suggesting that fusion partners should be proliferating cells or dendritic cells at a more immature stage . the resulting hybrid cells were shown to induce hepatoma - specific immunity and to protect against intrahepatically implanted small fragments of hepatoma cells when injected , unirradiated , in syngeneic rats . cd8α + , but not cd8α − , dendritic cells sensitize t helper - 1 type cells in vivo since their discovery in 1973 , dendritic cells have gained increasing interest from immunologists , since they appear to be the adjuvant of the immune system in vivo . dc are motile and efficiently cluster with t cells , are widely distributed in tissues , carry antigens that are administered intradermally and intravenously , and circulate through lymph and blood probably in route to lymphoid organs ( for review , see steinman , r . m ., pack , m . and k . inaba . 1997 . immunological reviews , 156 : 25 - 37 ). a new population of dendritic cells has been recently discovered that appears to display opposite properties in vitro , murine dendritic cells consist of both conventional cd8α − and cd8α + cells . cd8α + dc appear to express fasl , and through activation with fas on activated t cells induce their death by apoptosis in vitro ( vremec , d ., m . zorbas , r . scollay , d . j . saunders , c . f . ardavin , l . wu and k . shortman , 1992 . j . exp . med . 176 : 47 - 58 ; süss g . and k . shortman , 1996 , j . exp . med . 183 : 1789 - 1796 ). the cd8α + population resembles the population of dendritic cells in the thymus that plays a role in negative selection of thymocytes . we have shown previously ( sornasse , t ., v . flamand , g . de becker , h . bazin , f . tielemans , k . thielemans , j . urbain , o . leo and m . moser , 1992j . exp . med . 175 : 15 - 21 ; de smedt , t ., m . van mechelen , g . de becker , j . urbain , o . leo and m . moser , 1997 , eur . j . immunol . 27 : 1229 - 1235 ) that a single injection of antigen - pulsed splenic dc in syngeneic mice induced the activation of t helper cells of type 1 ( secreting interferon - γ and il - 2 ) and type 2 ( producing il - 4 , il - 5 and il - 10 ). more recently , we compared the nature of the immune response induced in recipients injected with antigen - pulsed cd8α − or cd8α + dendritic cells . both subsets of dendritic cells were purified as follows : mild collagenase ( clsiii ; worthington biochemical corp ., freehold , n . j .) digestion for 25 min at room temperature and edta treatment were applied to release dc from murine spleen fragments . spleen cells were washed in ca ++ - free hbss medium containing edta and further separated into low and high density fractions on a nycodenz gradient ( nycomed pharma as , oslo , norway ). low density cells were cultured during 2 h in rpmi containing 2 % hy ultroser ( a serum - free medium supplement purchased from gibco brl , merelbeke , belgium ) and 50 μg / ml of gm - csf . the non - adherent cells were removed by vigorous pipetting . adherent cells were cultured overnight in the same medium with or without addition of antigen ( keyhole limpet hemocyanin , klh , 50 μg / ml ). dendritic cells were further separated into cd8α + and cd8α − on a minimacs column using anti - cd8α - coupled microbeads , according to the manufacturer &# 39 ; s recommendations ( miltenyi biotec gmbh , bergisch - gladbach , germany ) and washed in pbs ( phosphate buffered saline ), 3 × 10 5 cells in 50 - 100 μl were injected into the footpads of syngeneic mice . 5 days later , draining lymph nodes were harvested and unseparated lymph node cells were cultured in 2 % hy ultroser - containing rpmi in the presence of serial dilutions of klh . the proliferation was measured as thymidine incorporation during the last 12 - 16 h of the 2 - day culture . culture supernatants were assayed for interleukin - 2 after 24 h and for interferon - γ after 96 h of incubation . culture supernatants were assayed for il - 2 content by a standard elisa . interferon - γ was quantitated by two - site elisa using mab f1 and db - 1 , as previously described ( t . de smedt , et al . 1997 . eur . j . immunol . 27 : 1229 - 1235 ). the data in fig1 show that both subsets of dendritic cells , pulsed in vitro with klh , sensitized antigen - specific t cells in vivo , as assessed by proliferation upon antigen restimulation in culture . controls included untreated mice ( nt ) and mice that received unseparated dendritic cells ( cd8α +/− ). lymph node cells from untreated mice do not proliferate upon stimulation with klh in vitro . a similar pattern was observed for interleukin - 2 secretion . interestingly , cd8α + , but not cd8α − , dendritic cells induced the development of interferon - γ - secreting t cells ( th1 cells ) in the same conditions . lymph node cells from mice injected with unseparated dendritic cells secret intermediate levels of interleukin - 2 and interferon - γ . these data suggest that cd8α + dendritic cells strongly sensitive antigen - specific naive t cells and are required for th1 development in vivo . brichard ; 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