Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FLIPr) and its homologue FLIPr-like are potent FcγR antagonists that inhibit IgG-mediated effector functions.

To evade opsonophagocytosis, Staphylococcus aureus secretes various immunomodulatory molecules that interfere with effective opsonization by complement and/or IgG. Immune-evasion molecules targeting the phagocyte receptors for these opsonins have not been described. In this study, we demonstrate that S. aureus escapes from FcγR-mediated immunity by secreting a potent FcγR antagonist, FLIPr, or its homolog FLIPr-like. Both proteins were previously reported to function as formyl peptide receptor inhibitors. Binding of FLIPr was mainly restricted to FcγRII receptors, whereas FLIPr-like bound to different FcγR subclasses, and both competitively blocked IgG-ligand binding. They fully inhibited FcγR-mediated effector functions, including opsonophagocytosis and subsequent intracellular killing of S. aureus by neutrophils and Ab-dependent cellular cytotoxicity of tumor cells by both neutrophils and NK cells. In vivo, treatment of mice with FLIPr-like prevented the development of an immune complex-mediated FcγR-dependent Arthus reaction. This study reveals a novel immune-escape function for S. aureus-secreted proteins that may lead to the development of new therapeutic agents in FcγR-mediated diseases.

Mouse CD40-transfected cell lines cannot exhibit the binding and RANTES-stimulating activity of exogenous heat shock protein 70.

Here we demonstrate the inducible mouse Hsp72 binds markedly to lymphoid neoplastic macrophage-like P388D1 cells. To examine whether mouse CD40 can play a role in signaling exogenously administered HSP70 in a fashion similar to that of human CD40, we established mouse CD40-transfectants of both human 293 cells and murine-pro-B cell line Ba/F3. A small portion of mouse CD40 expressed on 293-derived transfectants was the mature form with a signal-transducible C-terminal domain, whereas a majority of expressed antigen showed the molecular size smaller than we expect. Flow cytometry showed that mouse Hsp72, but neither its deletion variants nor the related Escherichia coli DnaK, bound to the 293-derived transfectants regardless of CD40 expression. CD40 molecules expressed on the transfectants showed the binding of soluble form of CD40L but this binding was not inhibited by excess amount of HSP70. CD40L, but not any HSP70 recombinant proteins, stimulated the production of chemokine RANTES in the transfectants. Furthermore, no RANTES production was induced by HSP70-RCMLA complex in the transfectants, although it binds to 293-derived cells in a CD40-independent manner. No interaction between mouse CD40 and HSP70 recombinant proteins was detected by using the Ba/F3-derived transfectants that express the mature form of mouse CD40. The present results imply that mouse CD40 expressed on the transfectants differs from its human homolog in the binding of exogenously administered HSP70.

[Effect of bone marrow mesenchymal stem cells on acute graft versus host disease and graft versus leukemia after allogeneic bone marrow transplantation].

To investigate the effect of bone marrow mesenchymal stem cells (BMMSC) on acute graft versus host disease (aGVHD) and graft versus leukemia (GVL) after allogeneic bone marrow transplantation (allo-BMT), both bone marrow cells and BMMSC obtained after three to four weeks of culture from donor mice were transplanted into the recipient mice injected with acute lymphocytic leukemia cells 5 days before, the control group was injected with bone marrow cells alone. The survial time after allo-BMT was recorded; the general manifestation and pathological changes of aGVHD in recipient mice were observed; the effects of BMMSC on the quatity of CD4(+) and CD8(+) T cell in vivo after allo-BMT were evaluated by flow cytometry; chimerism was detected by sex chromosome. The results showed BMMSC could increase obviously the survival time, and delay onset of aGVHD, BMMSC could decrease the amount of CD4(+) T cell and increase CD8(+) T cell in vivo. It is concluded that cotransplantation of bone marrow cells with BMMSC from the same donor mice has GVL effect. BMMSC can alleviate aGVHD and maintain GVL effect after allo-BMT.

Effect of testosterone on growth of P388 leukemia cell line in vivo and in vitro. Distribution of peripheral blood T lymphocytes and cell cycle progression.

In transplanted mice, the P388 tumor grew better in castrated than in non castrated (NC) mice. The proportion of CD8+ in the blood was more numerous in NC mice. The T cell subsets (CD4+ and CD8+) were also high in the mice with small tumor tissue (<10 mg). The correlation observed between the tumor weight and T cell subset in PBL and in the mice with small tumors could confirm the important intervention of CD4+ and CD8+ cells to inhibit growth of tumor. Depo-testosterone (DT) injection reduced strongly weight and tumor growth in mice. On top of that, DT administration induced a significant increase in the percentage of blood CD8+ cells in grafted mice. The effect of DT was studied on the cell cycle progression, in tumor tissue of P388 tumor bearing BDF1 mice and in P388 murine leukemia cell line in culture. The cell cycle analysis in tumor tissue showed that DT decreased both the cells in S phase and the proliferating leukemic cells, with accumulation of cells in G0/G1 phase. The testosterone can inhibit the proliferation of leukemic cells with a pharmacological dose (10(-7) M). This growth inhibition, dose and time dependent, was associated with cell cycle arrest; P388 cells accumulates in G0/G1 phase. We also observed a correlation between tumor weight and the percentage of cells in G0/G1 and the relative number of cells in proliferative state (S + G2/M). To conclude, our experiments reported that testosterone prevents the growth of tumor: indirectly by modulation of subsets T cells distribution and directly by the alteration of the cell cycle.

Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida.

BACKGROUND: We showed that fucoidan, extracted from dietary seaweed, could inhibit tumor growth. However, the mechanism of Mekabu (Sporophyll of Undaria pinnatifida) fucoidan antitumor activity and how it enhances the immune response remains unknown. MATERIALS AND METHODS: We examined the effect of Mekabu fucoidan in P-388 tumor-bearing mice and in T cell-mediated NK cell activity in normal mice. RESULTS: The survival of mice was prolonged when Mekabu fucoidan was administered for 4 days before tumor cell inoculation, compared with non-treated mice. Fucoidan significantly enhanced the cytolytic activity of NK cells and increased the amount of IFN-gamma produced by T cells up to about 2-fold compared with non-treated mice. CONCLUSION: The anti-tumor effect of Mekabu fucoidan appears to be mediated by IFN-gamma-activated NK cells.

Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing hybrid Yersinia type III proteins.

In the present study, we have investigated the possibility to engage the Yersinia outer protein E (YopE) as a carrier molecule for heterologous Ag delivery by the type III secretion system of Salmonella typhimurium. Defined secretion and translocation domains of YopE were fused to the immunodominant T cell Ags listeriolysin O and p60 of Listeria monocytogenes. In vitro experiments showed that S. typhimurium allows secretion and translocation of large hybrid YopE proteins in a type III-dependent fashion. Translocation and cytosolic delivery of these chimeric proteins into host cells, but not secretion into endosomal compartments, led to efficient MHC class I-restricted Ag presentation of listerial nonamer peptides. Mice orally vaccinated with a single dose of attenuated S. typhimurium expressing translocated hybrid YopE proteins revealed high numbers of IFN-gamma-producing cells reactive with listeriolysin O 91-99 or p60 217-225, respectively. This CD8 T cell response protected mice against a challenge with L. monocytogenes. In conclusion, these findings suggest that YopE is a versatile carrier molecule for type III-mediated foreign Ag delivery by Salmonella vaccine strains.

Cytokine production by macrophages in association with phagocytosis of etoposide-treated P388 cells in vitro and in vivo.

Chemotherapy and radiotherapy are performed for cancer patients with the hope that dying cancer cells are safely scavenged by phagocytic cells such as macrophages. In this study, we examined cytokine production by macrophages during and after the phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Etoposide caused apoptosis as early as 5 h after treatment, as assessed as to the exposure of phosphatidylserine, increase in membrane permeability and DNA ladder formation. Phagocytosis by phorbol myristate acetate (PMA)-treated THP-1 cells occurred marginally when P388 cells were treated with etoposide for 10 h, while it occurred significantly with P388 cells treated for 24 h, as evidenced by flow cytometry and confocal microscopy. PMA-treated THP-1 cells produced pro-inflammatory cytokines, such as interleukin (IL)-1alpha, IL-8 and macrophage migration inhibitory factor (MIF), but not anti-inflammatory cytokines among those tested at the mRNA level during and after the phagocytosis of apoptotic cells. IL-8 and MIF were also produced at the protein level, and the IL-8 production was dependent on cell-to-cell contact when the plasma membranes of apoptotic cells were intact enough not to leak one of the cytoplasmic enzymes, lactate dehydrogenase. In addition, etoposide-treated P388 cells induced neutrophil infiltration as well as MIP-2 production upon injection into the peritoneal cavity of either normal mice or mice with sterile peritonitis. When macrophages ingesting and/or binding apoptotic P388 cells were isolated from the mice with sterile peritonitis using a cell sorter, they were found to produce MIP-2 upon culture.

Early macrophage and cytokine response during the growth of immunogenic and non-immunogenic murine tumors.

Very little data exist on the mechanisms of innate immunity during the first days after syngeneic tumor inoculation. Nonspecific macrophage reaction precedes the development of specific immune response and is important for further tumor growth and stroma formation. We investigated two lymphoma cell lines of the same origin, differing in immunogenicity: non-immunogenic parental strain P388 and its highly immunogenic subline P388/adria. Early systemic inflammatory response resulted in the enhancement of nitric oxide (NO) and superoxide production by peritoneal macrophages which was at a maximum on the first day after s.c. tumor inoculation and was observed in mice bearing either of these tumors independently of immunogenicity. It was followed by a transient elevation of the serum levels of pro-inflammatory cytokines: TNF-alpha IL-6. In order to evaluate the role of inflammatory response, vaccinations with lethally irradiated lymphoma cells were performed. After two weekly injections, the mice were challenged s.c. with live tumor cells of the same subline. Effective vaccination with P388/adria lymphoma cells induced retardation of tumor growth in parallel with down-regulation of peritoneal macrophage activity and abrogation of serum cytokine release. Non-effective immunization with P388 cells influenced neither tumor growth nor macrophage functions and cytokine level. Thus, a positive correlation was found between down-regulation of the inflammatory response and inhibition of tumor growth. We suppose that, in efficiently immunized mice, special mechanisms exist which are responsible for down-regulation of the inflammatory reaction. Macrophage products may facilitate tumor cell survival by preventing apoptosis or participate in the activation of tumor neoangiogenesis. Suppression of these activities may serve as an important tool for the inhibition of tumor growth at the early stages of malignant transformation.

Graft-versus-leukemia effect and graft-versus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation (allo-BMT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect. In the present study, we examined the contribution of cytotoxic effector mechanisms, which are mediated by tumor necrosis factor-alpha (TNF-alpha), Fas ligand (FasL), or perforin, to GVHD and GVL effect in a murine BMT model. Bone marrow cells plus spleen cells (BMS) from wild-type, FasL-defective, or perforin-deficient donors were transferred into lethally irradiated recipients in the parent (C57BL/6) to F1 (C57BL/6 x DBA/2) BMT model with or without prior inoculation of DBA/2 leukemia L1210 or P815 mast cytoma cells. The effect of anti-TNF-alpha antibody administration was also examined. Whereas the defect or blockade of each cytotoxic pathway could ameliorate lethal acute GVHD, the GVL effect was differentially affected. The wild-type BMS recipients died of acute GVHD within 50 days without residual leukemia cells. The FasL-defective BMS recipients showed 60%< survival over 80 days without acute GVHD or residual leukemia cells. Administration of anti-TNF-alpha antibody resulted in early leukemia relapse and the recipients died within 25 days with massive leukemia infiltration in the liver. The perforin-deficient BMS recipients died within 60 days with residual leukemia cells. These results suggest that blockade of the Fas/FasL pathway could be used for ameliorating GVHD without impairing GVL effect in allo-BMT.

Lipopolysaccharide-induced desensitization of junB gene expression in a mouse macrophage-like cell line, P388D1.

Treatment of a mouse macrophage cell line, P388D1, for 1 h with bacterial LPS caused a transient increase in the level of junB mRNA expression. These cells became refractory in terms of the junB gene response to exposure to a second round of LPS or lipid A, but not to PMA. The LPS-induced desensitized state was not due to the shortening of the half-life of junB mRNA, but was suggested, by nuclear run-on analysis, to be caused by reduction of junB gene transcription. Pretreating cells with herbimycin A, a tyrosine kinase inhibitor, substantially inhibited LPS-induced expression of junB mRNA and decreased tyrosine phosphorylation of 38- to 42-kDa proteins, which comigrated with p38 and p42 mitogen-activated protein (MAP) kinases. Parallel to down-regulation of junB mRNA expression, activation of the p38 MAP kinase was markedly reduced in LPS-tolerant cells, whereas activation of p42 MAP kinase was relatively constant. The specific p38 MAP kinase inhibitor, SB202190, potently inhibited LPS-induced junB mRNA expression. These results suggest that the LPS-induced desensitization of junB gene expression occurs at or upstream of the level of gene transcription and may be involved in a defective LPS-induced p38 MAP kinase pathway.

The microtubule depolymerizing drugs nocodazole and colchicine inhibit the uptake of Listeria monocytogenes by P388D1 macrophages.

Uptake of Listeria monocytogenes by different mammalian cells like macrophages and epithelial cells is dependent on functional actin filaments and hence susceptible to inhibition by cytochalasin. Here we show that phagocytic uptake of L. monocytogenes by P388D1 macrophages is also highly sensitive to treatment with the microtubule depolymerizing drugs nocodazole and colchicine. This sensitivity is cell type specific and much less pronounced in bone marrow-derived macrophages and Caco-2 epithelial cells. In contrast to nocodazole and colchicine, the microtubule stabilizing drug taxol has no significant effect on the uptake of L. monocytogenes by all three cell types tested.

Redundancy of C/EBP alpha, -beta, and -delta in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1.

C/EBP alpha, -beta, and -delta are members of the CCAAT/enhancer binding protein family of transcriptional regulators. All three of these factors are expressed by bone marrow-derived macrophages, with the DNA binding activity of C/EBP beta and -delta increased by treatment with LPS while that of C/EBP alpha is decreased. We have ectopically expressed each C/EBP protein in P388 lymphoblasts. The expression of any of these transcription factors is sufficient to confer the LPS-inducible expression of IL-6 and monocyte chemoattractant protein-1 to lymphoblasts, which normally lack C/EBP factors and do not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBP alpha, -beta, and -delta are redundant in regard to the expression of IL-6 and monocyte chemoattractant protein-1. Since C/EBP beta-deficient mice have been reported to be largely normal in their expression of proinflammatory cytokines, it is likely that the lack of C/EBP beta is compensated for by the induction of C/EBP delta upon LPS treatment.

Tyrosine phosphorylation is required for ehrlichial internalization and replication in P388D1 cells.

Replication of Ehrlichia risticii was inhibited in P388D1 cells when a protein tyrosine kinase inhibitor (genistein or herbimycin A) was added after internalization of the organism at 3 h postinfection. Upon addition of genistein at day 1, 2, 3, or 4 postinfection, further proliferation of E. risticii was prevented. The inhibition was reversible, since regrowth of E. risticii occurred upon the removal of genistein. Genistein prevented spreading of E. risticii from P388D1 cells to THP-1 cells. Genistein did not prevent binding of [35S]methionine-labeled E. risticii to P388D1 cells but did prevent internalization of [35S]methionine-labeled E. risticii. 14CO2 production from L-[14C]glutamine in Percoll density gradient-purified E. risticii was not inhibited by genistein or herbimycin A, which suggests that these reagents did not directly inhibit ehrlichial energy metabolism. Double indirect immunofluorescence labeling with antiphosphotyrosine antibody and anti-E. risticii antibody revealed colocalization of tyrosine phosphoproteins with ehrlichial inclusions. There was, however, no colocalization of phosphotyrosine with phagosomes containing 0.5-microm-diameter fluorescent beads. Western immunoblot analysis revealed that 52- and 54-kDa proteins were tyrosine phosphorylated only in infected cells and that phosphorylation of these two proteins was reduced when infected cells were treated with genistein for 6 h. These results suggest that protein tyrosine phosphorylation is specific and essential for ehrlichial internalization, replication, and spreading in macrophages but not for binding.

[Biotherapeutic efficacy of adoptive transfer of CD3AK cells in combination with cyclophosphamide and kappa-selenocarrageenan in P 388 leukemic mice].

OBJECTIVE: To study the antileukemia efficacy of a combination of adoptive transfer of CD3AK cells, cyclophosphamide (CTX), and kappa-selenocarrageenan (KSC) in P 388 leukemic mice. METHODS: CD3AK cells in normal DBA/2 murine splenocytes were induced with anti-CD3 antibody and low dose recombinant interleukin-2 (rIL-2). The P 388 murine leukemia model was induced by i.p. injection of P 388 cells into normal DBA/2 mice. The tumor-bearing mice were administrated with adoptively transferred CD3AK Cells and/or CTX and/or KSC. RESULTS: After tumor inoculation, the cellular immune function of P 388-bearing mice was supressed markedly. Adoptive transfer of CD3AK cells with low dose rIL-2 into the P 388 mice significantly enhanced the splenocyte proliferation (SP) induced by Con A, the NK cell activity and the splenocytic IL-2 production and prolonged their survival (45.19%); CTX (200 mg/kg) alone prolonged the survival of P 388-bearing mice (29.90%), but further decreased the immunodeficiency; combination of CTX and CD3AK passive transfer could prevent the reduction of SP, NK activity and IL-2 production in the leukemic mice and prolonged the survival (59.45%), combination of KSC and adoptively transfected CD2AK cells and/or CTX had a much better therapeutic efficacy for P 388 murine leukemia, 12.50%-75.00% of the leukemic mice were cured. CONCLUSION: KSC is a hopeful biological response modifier in cancer biotherapy, and tumor killing effector cells and chemotherapy plus BRM might be a promising candidate for human leukemia biotherapy.

Identification, molecular cloning, biologic properties, and tissue distribution of a novel isoform of murine low-affinity IgG receptor homologous to human Fc gamma RIIB1.

A cryptic splice donor site in the first intracytoplasmic (IC) exon of the murine Fc gamma RIIB gene generates a previously unknown Fc gamma RIIB isoform. Three membrane Fc gamma RIIB polypeptides of 37, 32, and 30 kDa were immunoprecipitated by Fc gamma RIIB-specific Abs, and three Fc gamma RIIB cDNAs of 1071, 987, and 930 bp were amplified by reverse transcriptase-PCR with Fc gamma RIIB-specific oligonucleotides from the mastocytoma cells P815. The 1071-bp cDNA contains all sequences of IC exons and encodes the 37-kDa Fc gamma RIIB1 isoform. The 930-bp cDNA lacks sequences of the first IC exon and encodes the 30-kDa Fc gamma RIIB2 isoform. The 987-bp cDNA has an 84-nucleotide deletion of the first IC exon 3' sequences. When stably transfected in the lymphoma B cells IIA1.6, or in the mast cells RBL-2H3, this cDNA encoded 32-kDa Fc gamma RIIB whose biologic properties were undistinguishable from those of Fc gamma RIIB1: they inhibited B cell activation when coaggregated to B cell receptors and capped when aggregated at 37 degrees C, but failed to mediate endocytosis or phagocytosis. Sequences responsible for capping and inhibition of internalization, previously assigned to sequences encoded by the first IC exon, can thus be mapped in the 19 N-terminal residues. These residues are highly conserved in human Fc gamma RIIB1. The 87-bp first IC exon of the human gene ends by a single splice site in the downstream intron and encodes a 19-amino acid insertion. The 32-kDa Fc gamma RIIB is the murine homologue of human Fc gamma RIIB1. We propose to name it Fc gamma RIIB1'. Fc gamma RIIB1' was expressed in myeloid and lymphoid cell lines, in normal spleen cells, and in resting or LPS-activated B cells.

Involvement of macrophages and cytokines into rejection mechanism of the drug-resistant and immunogenic murine lymphoma P388/adria.

Macrophages and their products may exert either inhibitory or stimulatory effects on malignant cells,thus preventing or supporting tumor growth, however, the mechanisms of this interaction are not fully understood. It was the aim of the present study to elucidate the role of macrophage activation during the growth and rejection of highly immunogenic murine leukemia P388/adria cell line which was made resistant by suboptimal treatment of mice with adriablastin during the serial passaging of parental P388 cells. The functional activity of peritoneal macrophages and the serum level of cytokines IL-1 beta, IL-6 and TNF-alpha were studied in different groups of mice. Mice from group 1 (control) received saline. Mice from group 2 (tumor bearers) with fast subcutaneous (s.c) 100% tumor growth were compared with animals from group 3 that had been twice previously immunized with lethally irradiated P388/adria cells and later inoculated with viable tumor cells. Tumors grew in only 25% of group 3 animals with a significant delay. The activity of peritoneal macrophages was studied by NO2- production and the NBT-test. Both tests revealed the early high systemic activation of macrophages in group 2. This coincided with the elevation of serum TNF-alpha and IL-6 levels. This effect was not dependent on whether alive or lethally irradiated tumor cells were inoculated. The NO2- production by peritoneal macrophages correlated well with the dynamics of serum cytokine levels while the NBT-test did not. Studies on group 3 showed total abrogation of early macrophage and cytokine reactions. The production of inhibitory factors by macrophages in previously immunized mice is suggested. The fact that the early activation of macrophages and increase of serum levels of proinflammatory cytokines occurred in animals with fast growing tumors, which was decreased or absent in animals with tumor delay or rejections, allows us to suppose that this reaction plays more a supporting than a protecting role for tumor growth.

Glucocorticoid-mediated immunomodulation: hydrocortisone enhances immunosuppressive endogenous retroviral protein (p15E) expression in mouse immune cells.

To define glucocorticoid (GC)-regulated genes contributing to the anti-inflammatory and immunosuppressive effects of GC, previous work from our laboratory revealed up-regulation of transcripts from endogenous type B mouse mammary tumour virus (Mtv) and type C murine leukaemia virus (Emv) loci by high dose GC treatment of P388D1 macrophage-like cells. This study demonstrates enhancement of expression from Mtv and Emv loci in P388D1 cells by more physiological hydrocortisone concentrations (1 microM), and shows direct transcriptional mode of regulation by blocking GC-mediated signal transduction at different levels. Furthermore, we found up-regulation of Emv mRNA steady-state levels in murine lymphoid lineage cells (T-like EL4 and BW5147 cells; B-like X63 cells) upon GC treatment. The Emv transcripts shown by us to be GC-up-regulated encode for the transmembrane envelope protein TM/p15E which is highly conserved in several retroviruses. TM/p15E and the p15E-like products found in humans exert immunosuppressive effects in different test systems. Thus, our findings raise the possibility that immunomodulation by GC might be mediated in part by enhanced expression of p15E(-like) products.

[A morphofunctional evaluation of the immunomodulating action of nitrosomethylurea]. / Morfofunktsional'naia otsenka immunomoduliruiushchego deistviia nitrozometilmocheviny.

The thymuses and spleens from healthy and tumor-bearing (leukemia P 388 and Lewis' carcinoma) were morphometrically studied after a single administration of the antitumor agent nitrosomethylurea in doses of 96 and 4 mg/kg. When used both in the high and low doses, nitrosomethylurea had an immunomodulating effect, causing the activation of cell-mediated immunity in healthy and tumor-bearing (in early malignancy) animals when given in the small dose.

Characterization of four drug-resistant P388 sublines: resistance/sensitivity in vivo, resistance-and proliferation-markers, immunogenicity.

It was the aim of this study to compare drug-resistant sublines of the murine P388 in relation to resistance markers, the resistant phenotype and immunogenicity. Resistance to drugs either belonging to the MDR type (Doxorubicin, Vincristine, Mitoxantrone) or to the non-MDR type (Methotrexate) was generated in vivo in order to mimic the clinical situation. All resistant sublines expressed the mdr1 gene and the p-glycoprotein determined on m-RNA level or immunohistochemically, while no expression was registered in the parent P388. The rhodamine 123 fluorescence as marker for the energy dependent drug efflux pump was decreased only in the MDR-sublines, while the parent P388 and the Methotrexate-resistant line retained 100% or 90% of the dye, respectively. This indicates that the rhodamine efflux is a more function-related marker for MDR than the mdr1 gene and the pgp. The in vivo characterization of the sublines as regards their sensitivity to cytostatics revealed a clear-cut cross-resistance to MDR drugs in the MDR-lines, while the Methotrexate resistant subline was only cross-resistant to Cytarabine. In each resistant subline collateral sensitivity to certain but different cytostatics was observed. Experiments to overcome resistance by concomitant treatment with the modulators Nifedipine, Verapamil, Cyclosporin A and Chloroquin led to only limited success. The sublines P388/Mitox, P388/Vinc and P388/MTX developed immunogenicity which was never registered in the original P388. Vaccination with lethally irradiated drug-resistant cells resulted in a substantial rejection of viable tumor cells of the same line. With the P388/Mitox and P388/Vinc also an over-cross immunization was possible. This generation of immunogenicity as a concomitant characteristic of resistance should be considered as therapeutic potential also in the treatment of clinical cancer.