Development of a prognostic risk model for clear cell renal cell carcinoma by systematic evaluation of DNA methylation markers.

BACKGROUND: Current risk models for renal cell carcinoma (RCC) based on clinicopathological factors are sub-optimal in accurately identifying high-risk patients. Here, we perform a head-to-head comparison of previously published DNA methylation markers and propose a potential prognostic model for clear cell RCC (ccRCC). PATIENTS AND METHODS: Promoter methylation of PCDH8, BNC1, SCUBE3, GREM1, LAD1, NEFH, RASSF1A, GATA5, SFRP1, CDO1, and NEURL was determined by nested methylation-specific PCR. To identify clinically relevant methylated regions, The Cancer Genome Atlas (TCGA) was used to guide primer design. Formalin-fixed paraffin-embedded (FFPE) tissue samples from 336 non-metastatic ccRCC patients from the prospective Netherlands Cohort Study (NLCS) were used to develop a Cox proportional hazards model using stepwise backward elimination and bootstrapping to correct for optimism. For validation purposes, FFPE ccRCC tissue of 64 patients from the University Hospitals Leuven and a series of 232 cases from The Cancer Genome Atlas (TCGA) were used. RESULTS: Methylation of GREM1, GATA5, LAD1, NEFH, NEURL, and SFRP1 was associated with poor ccRCC-specific survival, independent of age, sex, tumor size, TNM stage or tumor grade. Moreover, the association between GREM1, NEFH, and NEURL methylation and outcome was shown to be dependent on the genomic region. A prognostic biomarker model containing GREM1, GATA5, LAD1, NEFH and NEURL methylation in combination with clinicopathological characteristics, performed better compared to the model with clinicopathological characteristics only (clinical model), in both the NLCS and the validation population with a c-statistic of 0.71 versus 0.65 and a c-statistic of 0.95 versus 0.86 consecutively. However, the biomarker model had limited added prognostic value in the TCGA series with a c-statistic of 0.76 versus 0.75 for the clinical model. CONCLUSION: In this study we performed a head-to-head comparison of potential prognostic methylation markers for ccRCC using a novel approach to guide primers design which utilizes the optimal location for measuring DNA methylation. Using this approach, we identified five methylation markers that potentially show prognostic value in addition to currently known clinicopathological factors.

A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis.

BACKGROUND: We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS: Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS: MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S): This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

Magnesium intake and colorectal cancer risk in the Netherlands Cohort Study.

Energy-adjusted magnesium intake was nonsignificantly inversely related to risk of colorectal cancer (n=2328) in the Netherlands Cohort Study on Diet and Cancer that started in 1986 (n=58 279 men and 62 573 women). Statistically significant inverse trends in risk were observed in overweight subjects for colon and proximal colon cancer across increasing quintiles of magnesium uptake (P-trend, 0.05 and 0.02, respectively). Although an overall protective effect was not afforded, our results suggest an effect of magnesium in overweight subjects, possibly through decreasing insulin resistance.

Influence of SERTPR and STin2 in the serotonin transporter gene on the effect of selective serotonin reuptake inhibitors in depression: a systematic review.

Large differences in clinical response to selective serotonin reuptake inhibitors (SSRIs) are observed in depressive patients with different genotypes. Quantification of these differences is needed to decide if genetic testing prior to antidepressant treatment is useful. We conducted a systematic review of the literature on the influence of polymorphisms in the serotonin transporter gene (SERTPR (or 5-HTTLPR) and STin2) on SSRI response. Studies were identified by the use of MEDLINE, EmBase and PsycINFO, references of articles, reviews and information from pharmaceutical companies. Nine studies assessing the influence of SERTPR or STin2 on treatment response were included. Outcome was expressed as the percentage of decrease in depression score (HAM-D or MADRS) or as the percentage of responders (> or =50% reduction on the depression scale). Both study methodologies and study outcomes showed large heterogeneity. Weighted mean decreases in depression score for patients with the s/s, s/l and l/l genotypes were 35.4, 46.3 and 48.0% at week 4, respectively, and 53.9, 54.6 and 48.3% at week 6. Among Caucasian patients, both mean decrease in depression score and response rate were lowest in the s/s group, while among Asian patients, results were inconsistent. Weighted response rates were 36.1% for the 10/12 genotype of the STin2 polymorphism and 80.7% for the 12/12 genotype (chi2=27.8, P<0.001) (only Asians). The available evidence points to a less favourable response to SSRI treatment among Caucasian patients with the SERTPR s/s genotype and among (Asian) patients with the STin2 10/12 genotype. In view of the scarcity and heterogeneity of the studies, however, current information is insufficiently reliable as a basis for implementing genetic testing in the diagnostic work-up of the depressive patient.

Interaction between smoking, GSTM1 deletion and colorectal cancer: results from the GSEC study.

Cigarette smoking has inconsistently been associated with an increased risk of colorectal cancer. One of the enzymes responsible for the detoxification of the carcinogenic compounds present in tobacco smoke is glutathione S-transferase-mu (GST-mu). The gene that codes for this enzyme is GSTM1. In this study, we evaluated the associations and interaction between GSTM1 deletion, smoking behaviour and the development of colorectal cancer. We performed a pooled analysis within the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). We selected six studies on colorectal cancer, including 1130 cases and 2519 controls, and restricted our analyses to Caucasians because the number of patients from other races was too limited. In addition we performed a meta-analysis including the studies from the GSEC database and other studies identified on MEDLINE on the same subject. The prevalence of the GSTM1 null genotype was within the range reported in other studies: 51.8% of the cases had the GSTM1 null genotype versus 56.6% of the controls. No significant association between the GSTM1 null genotype and colorectal cancer was found (odds ratio 0.92, 95% confidence interval 0.73-1.14). Our results suggest a possible positive association between lack of the GST-mu enzyme and colorectal cancer for non-smoking women (odds ratio 1.47, 95% confidence interval 0.80-2.70). There was no interaction between the effects of smoking and GSTM1 genotype on colorectal cancer risk in men and women (chi2=0.007, p=0.97). Our findings do not support an association between the GSTM1 null genotype and colorectal cancer. In addition, we did not find any modification of the smoking-induced colorectal cancer risk by GSTM1 genotype

Flt-3 ligand (FL) drives differentiation of rat bone marrow-derived dendritic cells expressing OX62 and/or CD161 (NKR-P1).

Bone marrow-derived dendritic cells (DC) of the rat have not been as well characterized as those from the mouse. Here, large quantities of bone marrow-derived rat DC were generated when Flt-3 ligand (FL) was used as an adjunct to granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). These cells displayed a typical DC phenotype, expressing MHC class II, CD54, CD80, CD86, and CD11b/c. These DC also uniformly expressed low levels of CD161 and expressed OX62 in a bimodal distribution. Few cells were recovered from cultures grown without FL, and they failed to express OX62 or CD161. The DC generated with FL were more potent antigen-presenting cells in mixed lymphocyte cultures than cells grown without FL, and among FL-derived cells, the OX62+ cells were slightly more stimulatory than OX62- cells. Thus, FL is a useful cytokine for obtaining large quantities of functional rat DC subsets in vitro.

Immunization with an antigen identified by cytokine tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo.

We have reported previously that s.c. immunization of rats with IL-4 transduced 9L gliosarcoma cells (9L-IL-4) induced a potent antitumor immunity against intracranial, parental 9L tumors. Subcutaneous implantation of 9L-IL-4 influenced the systemic humoral response, which was demonstrated by Th2-type isotype-switching and the induction of cellular immune responses, which played a critical role in the rejection of tumors. Serological analyses of recombinant cDNA expression libraries (SEREX), has recently emerged as a powerful method for serological identification of tumor-associated antigens (TAAs) and/or tumor rejection antigens (TRAs). Because IL-4 is known to activate B cells and to promote humoral responses, and inasmuch as induction of humoral responses by central nervous system tumors has been reported to be minimal, we investigated whether the induction of a potent humoral immune response against 9L TAAs or TRAs in rats immunized s.c. with 9L-IL4 could be demonstrated. Screening of 5 x 10(5) independent clones of 9L-expression cDNA library for the presence of reactive antibodies in the serum from a 91-IL-4 immunized rat led to the identification of three different TAAs. One 9L TAA (clone 29) was demonstrated to be calcyclin, a member of the S-100 family of calcium-binding proteins. The second 9L TAA (clone 37) was demonstrated to be the rat homologue of the J6B7 mouse immunomodulatory molecule. The third TAA (clones 158 and 171) was determined to be the rat homologue of the mouse Id-associated protein 1 (MIDA1), a DNA-binding, protein-associated protein. Northern blotting demonstrated that message for calcyclin was overexpressed in 9L cells. Message encoding MIDA1 was highly expressed in parental 9L cells and thymus and, to a lesser degree, in testis, suggesting that MIDA1 was comparable with the cancer/testis category of TAAs. Sera obtained from animals bearing 9L-IL-4 were found to have a higher a frequency and titer of antibodies to these antigens when compared with sera obtained from rats bearing sham-transduced 9L (9L-neo) cells. To determine whether immunization with these TAAs induced antitumor immunity, animals were immunized by intradermal injection with expression plasmids encoding calcyclin or MIDA1. Subsequent challenge of rats with parental 9L resulted in significant suppression of tumor growth in animals immunized with MIDA1, but not with calcyclin. These results indicate that MIDA1 is an effective 9L TRA and will be useful for the investigation of specific antitumor immunity in this glioma model. Furthermore, these results suggest that this approach, termed "cytokine-assisted SEREX (CAS)," may serve as an effective strategy for identification of TRAs for in animal-glioma models of cytokine gene therapy, and potentially in humans undergoing cytokine gene therapy protocols as well.

Role of NK cells in adoptive immunotherapy of metastatic colorectal cancer in a syngeneic rat model.

This article reviews our immunotherapy research with natural killer (NK) cells in a syngeneic rat colorectal cancer liver and lung metastasis model. Using adoptive transfer of interleukin (IL)-2-activated NK cells, NK cells were shown to selectively infiltrate the tumors. More NK cells were found in tumors when the NK cells were directly injected into tumor-draining blood vessels than when the cells were injected in systemic blood vessels. Under optimal conditions, a limited, though significant, effect of adoptively transferred NK cells on tumor growth was shown. We observed that both endogenous and adoptively transferred NK cells were predominantly present in the stroma surrounding the tumor cell nodules. It is possible that they did not penetrate the nodules containing the tumor cells because of the presence of a basal membrane-like structure around these nodules. Adoptively transferred NK cells may initiate elimination of tumor cells by activating other effector cells, whereas some may eliminate tumor cells by direct cell-cell contact. A diverse array of molecules was shown to be involved in this process. CD45 on NK cells was found to be important in initiating the lysis-inhibitory signal upon binding of 'self' major histocompatibility complex (MHC) class I on potential target cells. Our results indicate that NK-cell cancer therapy is still promising and needs improvement.

NK cells modulate MHC class I expression on tumor cells and their susceptibility to lysis.

Cytotoxicity and production of cytokines are two important functions of NK cells. These two different NK functions were studied in a syngeneic rat model in relation to MHC class I expression. We focussed on the mechanism by which NK cells modulate MHC class I expression on target cells and how this interferes with NK cell-mediated lysis. Using transfection experiments an inhibitory role on NK cell cytotoxicity for expression of target cells of RT1.A, rat MHC class I, was found. Co-culturing syngeneic tumor cells and NK cells resulted in enhanced MHC class I expression on the surviving tumor cell fraction, which was less susceptible to NK lysis. Increased tumor cell MHC class I was due to production of a soluble factor by NK cells, most likely interferon gamma. The regulatory function of NK cells shows here, that the enhancing of MHC class I expression on tumor cells in vitro and in vivo, results in downregulation of their target cell killing, but at the same time may facilitate the cytotoxic T cell function.

Cytokine gene therapy of gliomas: induction of reactive CD4+ T cells by interleukin-4-transfected 9L gliosarcoma is essential for protective immunity.

Tumor cells genetically modified to secrete cytokines stimulate potent immune responses against peripheral and central nervous system tumors; however, variable results on the efficacy of this strategy for therapeutic intervention against established intracranial neoplasia have been reported. We have found that vaccination with rat 9L gliosarcoma cells expressing interleukin 4 (9LmIL4) induced a specific, protective, immune response against rechallenge with parental 9L tumors. In naive rats, sham-transfected 9L (9Lneo) tumors and 9LmIL4 tumors grew at comparable rates for 12-14 days, and then 9LmIL4 tumors regressed. After regression of 9LmIL4 tumors, rats were resistant to rechallenge with parental 9L cells. To investigate the mechanism(s) responsible for 9LmIL4-induced immunity, the phenotype and function of tumor-infiltrating lymphocytes (TILs) in 9Lneo and 9LmIL4 tumors were compared. In flow cytometric analyses, it was determined that CD4+ T cells were the predominant cell type in both 9Lneo and 9LmIL4 tumors at day 10. However, at the onset of regression (day 14), 9LmIL4 tumors were infiltrated predominantly by CD8+ T cells. To investigate functional aspects of the anti-9L tumor responses, we assessed the capacity of 9LmIL4 TILs to mediate specific lytic function or production of cytokines. In response to parental 9L, TILs isolated from day 14 9LmIL4 tumors were demonstrated to produce substantially greater amounts of IFN-gamma than did TILs from 9Lneo tumors. Although freshly isolated TILs from 9LmIL4 or control tumors did not lyse 9L cells in 51Cr-release cytotoxicity assays, specific cytotoxicity was demonstrable using TILs from day 14 9LmIL4 or splenocytes from 9LmIL4-bearing rats after their restimulation for 5 days with parental 9L tumor cells in vitro. Antibody blocking studies demonstrated that cytokine production and lytic activity by TILs, or splenocytes from 9LmIL4-immunized rats, were mediated in a T-cell receptor-dependent fashion. Because interleukin-4 also promotes humoral responses, quantity and isotype of immunoglobulins in sera from 9Lneo or 9LmIL4-immunized rats were compared. The amount of IgG1 antibodies was significantly increased in sera from 9LmIL4-immunized rats compared to sera from 9Lneo-bearing rats. Experiments using sublethally irradiated, naive rats adoptively transferred with splenocytes and/or sera from 9LmIL4-immunized or naive rats demonstrated that immune cells, with or without immune sera, protected recipients from challenge with parental 9L. Immune sera provided no protection when given with lymphocytes from naive rats, and it did not enhance protection against parental 9L when given in conjunction with lymphocytes for 9LmIL4-immunized rats. In additional adoptive transfer experiments, an essential role for CD4+ T cells in immunity was observed because their depletion from among splenocytes of 9LmIL4-immunized rats eliminated the protective effective against 9L, whereas depletion of CD8+ cells resulted in a more limited effect on protection against 9L. These data suggest that strategies for inducing systemic, long-term tumor-specific reactivity among CD4+ T cells will be critical for the development of immunotherapy of gliomas.

Effective cytokine gene therapy against an intracranial glioma using a retrovirally transduced IL-4 plus HSVtk tumor vaccine.

To explore the potential for molecular immunotherapies in the treatment of malignant gliomas, we evaluated the efficacy of subcutaneous tumor cell vaccines in the treatment of intracranial 9L tumors, using 9L gliosarcoma cell lines stably transduced with the murine interleukin-4 cDNA (9L-IL4), the herpes simplex virus-thymidine kinase cDNA (9L-Tk) or both (9L-IL4-Tk). The expression of multiple genes from a single transcript was achieved by incorporating internal ribosomal entry site (IRES) cassettes in the retroviral constructs. Subcutaneous immunization of rats with nonirradiated 9L-IL4 cells or 9L-IL4-Tk cells followed by treatment with ganciclovir (GCV) completely protected the animals from a subsequent intracranial challenge with wild-type 9L cells. In contrast, only 50% of animals immunized with 9L-Tk cells and 0% of 9L-neo immunized animals rejected the same challenge with wild-type 9L. More importantly, treatment of established (day 3) intracranial 9L tumors with genetically engineered tumor cells resulted in long-term survival (> 100 days) for 25-43% of 9L-IL4-Tk immunized animals and for 27% of nonirradiated 9L-IL4 immunized animals. In striking contrast, no 9L-Tk, 9L-neo or irradiated 9L-IL4 immunized animals survived for more than 33 days. As a marker of a cellular immune response, splenocytes from nonirradiated 9L-IL4, 9L-Tk or 9L-IL4-Tk immunized animals produced interferon-gamma (IFN-gamma) in greater amounts than those from 9L-neo immunized or Hank's balanced salts solution (HBSS) treated animals when stimulated with wild-type 9L in vitro. Our findings support the use of tumor cell vaccines expressing the IL-4 and HSVtk genes for the treatment of malignant gliomas.

The regulatory role of CD45 on rat NK cells in target cell lysis.

To investigate the role of CD45 in rat NK cell function, we developed new mAbs directed against rat CD45. mAb ANK12 binds to a high molecular isoform of CD45 and mAb ANK74 binds to the common part on all known CD45 isoforms, as has been described for the anti-rat CD45 mAb OX1. The ability of these mAbs to affect NK cell-mediated lysis was tested using the Fc receptor-positive target cell line P815. mAb ANK12 was found to significantly enhance the lysis of P815, whereas ANK74 and the anti-CD45 mAb OX1 did not. In addition, cross-linking of the CD45 isoform by ANK12 induced tyrosine phosphorylation of specific proteins in NK cells. Subsequently, the involvement of CD45 in the negative signaling after "self" MHC class I recognition by rat NK cells was investigated. The anti-CD45 mAbs were found to affect NK cell-mediated lysis of syngeneic tumor cell lines, depending upon the expression level of MHC class I on target cells. mAbs ANK74 and OX1 only inhibited lysis of the syngeneic tumor cell lines that expressed low levels of MHC class I. Furthermore, both mAbs caused an inhibition of NK cell-mediated lysis of these tumor cell lines when MHC class I molecules on the tumor cell lines were masked by an Ab. These results suggest that CD45 regulates the inhibitory signal pathway after self MHC class I recognition, supposedly by dephosphorylation of proteins.

Bone marrow-derived dendritic cells pulsed with a tumor-specific peptide elicit effective anti-tumor immunity against intracranial neoplasms.

Although the central nervous system (CNS) is often regarded as an immunologically privileged site, it is well established that specific CNS immunoreactivity can be generated through peripheral vaccination with CNS antigens. Dendritic cells (DC) are potent antigen presenting cells of hematopoietic origin that have emerged as a promising tool for cancer immunotherapy capable of evoking significant anti-tumor immunity when pulsed with tumor-associated peptides. To explore a role for DC-based immunization strategies for the treatment of CNS tumors, we developed a brain tumor model using the C3 sarcoma cell line which expresses the tumor-specific, major histocompatibility complex (MHC) class I-restricted peptide epitope E7(49-57). Syngeneic C57Bl/6 mice receiving intravenous (i.v.) injections of bone marrow-derived DCs pulsed with E7 peptide were effectively protected against a subsequent intracerebral challenge with C3 tumor cells. More importantly, this systemic immunization strategy was effective in a therapy model as 67% of animals (10 of 15) with established (day 7) intracerebral C3 tumors treated with 3 weekly injections of E7 peptide-pulsed DCs achieved a long-term survival (>90 days) while no control animals survived beyond day 41. In vivo depletion of CD8+ cells, but not CD4+ or asialo-GM1+ cells, abrogated the efficacy of E7 peptide-pulsed DC therapy of established tumors, indicating a pivotal role of specific CD8+ T-cell responses in mediating the anti-tumor effect. Our findings support the hypothesis that effective CNS anti-tumor immunoreactivity can be generated with DC-based tumor vaccines.

Novel monoclonal antibodies against membrane structures that are preferentially expressed on IL-2-activated rat NK cells.

Interleukin-2 (IL-2)-activated natural killer (NK) cells are known to mediate specific functions such as cytolytic activity and tumor infiltration more efficiently than nonactivated NK cells. To investigate whether these characteristics are associated with induction or up-regulation of expression of membrane structures after IL-2 activation, we selected four hybridomas (mAbs ANK44, ANK66, ANK7, and ANK123) derived from mice immunized with rat IL-2-activated NK cells and compared the expression of the epitopes recognized on IL-2-activated NK cells versus unstimulated NK cells. We found that ANK44-expression was induced after activation with IL-2. The antigens recognized by ANK66, ANK7, and ANK123 were expressed selectively on subsets of nonactivated NK cells. After activation with IL-2 the level of expression of these antigens was increased. Moreover, the majority of NK cells then expressed these antigens after IL-2 activation. Biochemical characterization of the membrane structures recognized on IL-2-activated NK cells showed that they have not previously been described. The precise function of these membrane structures is not yet known, however, the selective nature of their expression implies their involvement in the specific functions of IL-2-activated NK cells.

Characterization of three new membrane structures on rat NK cells which are involved in activation of the lytic machinery.

In this study, three membrane structures on rat NK cells which activate lysis of target cells were characterized. Furthermore, the role of adhesion molecules in this activation process, in particular the CD18-associated integrins, was investigated. Three rat NK-activation structures were identified which have not been previously described. These structures are apparently unique as they differed in molecular weight from known NK-activation structures. Cross-linking of these activation structures with specific mAbs and a Fc gamma R-positive tumor cell line (P815) resulted in enhanced killing of these target cells by NK cells. If the CD18-associated integrins were masked by the anti-CD18 mAb WT.3, the redirected killing of P815 was completely blocked. This indicates that the CD18-associated integrins play a crucial role in activation of NK cells. Furthermore, our results show that rat NK cells possess multiple activation structures.

The role of MHC class I expression in rat NK cell-mediated lysis of syngeneic tumor cells and virus-infected cells.

In this study the role of MHC class I antigen expression in rat natural killer (NK) cell-mediated lysis was investigated. Various rat tumor cell lines and two Adenovirus (Ad)-transformed rat cell lines were tested for their expression levels of total MHC class I and two MHC class I alleles, RT1.A and RT1.C, by flow cytometry. Their susceptibility to NK cell-mediated lysis in relation to MHC class I expression was determined by 51Cr release assays. IFN-gamma is know to increase the expression of MHC class I. Therefore target cell with and without prior IFN-gamma treatment were examined for MHC class I expression and its effect on NK lysis. An significant inverse exponential relationship was found. To investigate the effect of virus infection on MHC class I expression and target cell lysis by NK cells, rat embryonal fibroblasts (REF) were infected with cytomegalovirus (CMV) and used as target cells for NK cell-mediated lysis. Results showed that these virus-infected cells were less susceptible to NK lysis than non-infected cells. Moreover, the non-infected cells expressed less MHC class I than the infected cells, indicating that also in this case, there was an inverse correlation between MHC class I expression and susceptibility to lysis by NK cells. Subsequently, we showed that sorted subsets of predominantly CD8-positive and CD8-negative NK cells lysed a MHC class I-positive tumor cell line at the same level. This suggests that CD8 is not likely to participate as a receptor for MHC class I in NK cell-mediated lysis in a syngeneic rat model.

Rat interleukin-2-activated natural killer (A-NK) cell-mediated lysis is determined by the presence of CD18 on A-NK cells and the absence of major histocompatibility complex class I on target cells.

The precise mechanism by which target cells are recognized and subsequently lysed by interleukin-2-activated natural killer (A-NK) cells is poorly understood. In this study the role of major histocompatibility complex (MHC) class I and adhesion molecules in the recognition and lysis of tumor cells was investigated in a syngeneic Wag rat model. Preincubation of tumor cells with F(ab')2 fragments of anti-MHC class I monoclonal antibody (mAb) OX18 strongly enhanced the A-NK cell-mediated lysis. Also normal syngeneic cells such as T cells and A-NK cells became highly sensitive for lysis by A-NK cells after preincubation with mAb OX18. Two other mAb against MHC class I had no effect on lysis of target cells. These data indicate that masking of MHC class I on syngeneic tumor and normal cells by mAb OX18 is sufficient for A-NK cells to recognize target cells as non-self, resulting in lysis. In addition, we found that the presence of mAb against the beta 2 (CD18)-integrins blocked the lysis of all tumor cell lines by A-NK cells in 51Cr-release assays, also when target cells were preincubated with mAb OX18. Because of the absence of CD18 on most tumor cells we concluded that a CD18-associated integrin on A-NK cells is essential for lysis of target cells. These results show that in this syngeneic rat model CD18 on A-NK cells together with MHC class I on tumor cells determine A-NK cell-mediated lysis. Furthermore, we hypothesize that the anti-MHC class I OX18 recognizes an epitope on rat MHC class I which is, or is very close to, the restriction element determining A-NK cell-mediated lysis.

The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531.

In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 x CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of 51Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 x CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.