Tumour immunology, vaccination and escape strategies.

Our increasing knowledge of the mechanisms by which tumour cells escape immune effector cells is helping to establish new approaches to therapeutic vaccination against tumour development. One of the escape mechanisms used by tumour cells is the generation of multiple variants with different HLA phenotypes. These MHC class I phenotypic alterations play a key role in the tumour-host scenario, as they are crucial molecules for antigen presentation to T cells and modulation of natural killer (NK) cell activity. This review presents evidence indicating that tumours develop sophisticated MHC phenotypes that allow them to escape immune surveillance. We evaluate the importance of these alterations in terms of the potential development of therapeutic approaches to immune vaccination.

Phagocytosis of apoptotic cells assessed by flow cytometry using 7-Aminoactinomycin D.

BACKGROUND: Apoptotic cells are recognized specifically by macrophages and are cleared rapidly by phagocytosis. However, the recognition mechanisms involved in the clearance of apoptotic cells by macrophages are still not fully understood. Therefore, new methods must be designed to better our understanding of the mechanisms of interaction between macrophages and apoptotic cells. 7-Aminoactinomycin D (7-AAD) is a fluorescent DNA-binding stain usually used as a single agent to detect apoptotic cells by flow cytometry. We propose the use of 7-AAD-stained apoptotic cells as targets for a new flow cytometry phagocytosis assay. METHODS: Murine T-cell lymphoma YAC-1 cells were treated with etoposide to induce apoptosis. Etoposide-treated YAC-1 target cells were stained subsequently with 7-AAD and then coincubated with resident peritoneal macrophages to allow phagocytosis. The samples were analyzed by flow cytometry. Macrophages that had phagocytosed 7-AAD-stained apoptotic cells were identified by their bright red fluorescence and the resulting values were expressed as the percentage of cells. RESULTS: The phagocytic cells appeared as a distinct population characterized by bright fluorescence, which could not be detected in the negative controls. The effects of a phagocytic enhancer (interferon-gamma [IFN-gamma]) or inhibitor (incubation at 4 degrees C) were assessed accurately with this flow cytometric method. CONCLUSIONS: We describe the use of 7-AAD in an assay that is easy and quick to perform. This flow cytometric-based assay allows the quantification of phagocytosis of apoptotic cells by macrophages.

Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis.

BACKGROUND: Fluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry. METHODS: Suspensions of isopropyl alcohol-permeabilized Escherichia coli DH5alpha were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells. RESULTS: Results are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4 degrees C) or enhancer (gamma interferon [IFN- gamma] treatment) being accurately assessed with this assay. CONCLUSIONS: The method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells.

Suppression of splenic macrophage Candida albicans phagocytosis following in vivo depletion of natural killer cells in immunocompetent BALB/c mice and T-cell-deficient nude mice.

The resistance of mice to systemic infections caused by Candida albicans is associated with activated splenic macrophages. In addition, there is a correlation between natural killer (NK) cell activation and the resistance to systemic candidiasis. The present study was designed to clarify the role of NK cells in the control of splenic macrophage C. albicans phagocytosis by either depleting NK cells (anti-asialo GM(1) treatment) or maintaining them in an activated state (tilorone treatment) in both immunocompetent BALB/c mice and T-cell-deficient nude mice. The results of the in vitro phagocytosis assays were analyzed by flow cytometry and demonstrate the pivotal role of NK cells in controlling the capacity of splenic macrophages to phagocytose C. albicans. In summary, these data provide evidence that the NK cells are the main inducers of phagocytic activity of splenic macrophages and that they mediate the protection against C. albicans systemic infection.

Loss of natural killer cell activity after murine tumor transplantation appears as a consequence of dietary lipid administration.

It is generally assumed that unsaturated fatty acids are important biological mediators that may be applied in the treatment of patients suffering from autoimmune disorders, in the modulation of tumorigenic processes or in the induction or inhibition of apoptosis. In this study, mice divided into three groups were fed dietary lipids; olive oil (20% w/w), fish oil (20% w/w) or hydrogenated coconut oil (20% w/w) for four weeks. Then, survival percentage, natural killer (NK) cell activity and lipid peroxidation were measured after murine lymphoma (LSTRA) transplantation. Survival percentage showed a reduced resistance to LSTRA tumor in mice fed fish oil diet and generaly loss of NK cell activity was observed after lymphoma transplantation. Lipid peroxidation from mice fed dietary lipids was measured in the presence of both phospholipase and cyclooxygenase inhibitors. In general, the data contained in this paper point to a detrimental effect caused by dietary lipids in mice transplanted with a lymphoma.

The use of 7-amino-actinomycin D in the analysis of Candida albicans phagocytosis and opsonization.

We describe the use of 7-amino-actinomycin D (7AAD) to measure phagocytosis and the opsonizing capacity of serum. Heat-inactivated Candida albicans was previously stained with 7AAD and incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytic cells were identified by their bright red fluorescence. This is a rapid, reproducible and reliable one-step procedure and provides a means of evaluating low levels of phagocytosis.

Immunoselection by T lymphocytes generates repeated MHC class I-deficient metastatic tumor variants.

Alteration of MHC class I molecule expression is a widespread mechanism used by tumor cells to evade T cell responses. It has long been proposed that the origin of these MHC class I-negative or -deficient tumor variants is T cell immune selection. However, there are no experimental or clinical data to substantiate this hypothesis, and this issue is currently the subject of debate. Here we report that an H-2 class I-negative fibrosarcoma tumor clone generated MHC class I-negative spontaneous lung metastases in immunocompetent syngeneic BALB/c mice. Interestingly, the same B9 clone generated MHC class I-positive metastatic nodes, under basal conditions, in athymic nu/nu BALB/c mice. This phenomenon was observed in the metastatic nodules generated after a period of in vivo growth but not in the primary tumors growing locally in the footpad. These findings support the hypothesis that the H-2 phenotype of metastatic nodes is influenced by the T cell repertoire of the host, since in the absence of this T cell pressure (i.e., in nude mice) the metastatic nodes 'recovered' H-2 class I expression. In addition, 2 different phenotypes were found when the metastatic nodules obtained from immunocompetent mice were treated with IFN-gamma. One phenotype, present in 83% of the colonies, was characterized by resistance of the Ld molecule to IFN-gamma induction, due to a deletion involving the Ld gene. The second phenotype (17% of the colonies) was similar to the original B9 clone and was characterized by the response of K, D and L class I genes to IFN-gamma. These data provide evidence that the changes in MHC class I expression during tumor development might not be random but could be predictable.

Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice.

Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients. Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders. We wished to determine if this immune mechanism is involved in the specific response to C. albicans. Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity. In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis. We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis. Thus, it seems that the immunosurveillance of metastatic spread and the infection by C. albicans share some immune effector mechanisms, in particular activation of NK cells.

The HLA crossroad in tumor immunology.

It is generally accepted that human and experimental tumor cells can lose major histocompatibility complex (MHC) class I molecules. These human leukocyte antigen (HLA) losses are detected when the primary tumor breaks the basal membrane, invades the surrounding tissues, and starts to metastasize. These altered HLA class I phenotypes probably constitute the major tumor escape mechanism facing anti-tumor T-cell mediated responses. Thus, it is important to characterize these phenotypes in clinical tumor samples, analyze the mechanism(s) responsible for them, and counsel patients before and during peptide anti-cancer immunotherapy. The present paper summarizes the most relevant altered HLA class I phenotypes found in human tumor samples, indicates their frequency, and outlines the mechanisms implicated. This review also points out that the natural killer (NK) escape mechanism of HLA class I deficient cancer cells is yet to be defined. Knowledge accumulated to date reveals that HLA class I molecules are an important crossroad in tumor immunology.

The biological consequences of altered MHC class I expression in tumours.

The identification of different mechanisms by which tumours escape from the immune system has helped to evaluate the clinical relevance of a variety of phenotypic changes that occur during tumour development. Among them, changes in HLA class I expression play a leading role in the tumour-host environment since HLA class I molecules interact with T lymphocytes for antigen presentation and with NK cells for inhibition/activation of these immune effector cells. Our laboratory has proposed a classification of the altered HLA class I phenotypes frequently found in human tumours, into five major groups. This review focuses on the tumour phenotypes found in primary and metastatic lesions, the molecular mechanisms that give rise to each phenotype and the clinical implications of these findings.

Differential effect of protein-bound polysaccharide (PSK) on survival of experimental murine tumors.

The effect of protein-bound polysaccharide (PSK) on the survival of BALB/c and C57BL/6 mice after intravenous injections of syngeneic murine sarcomas (GR9.B9 and Meth-A), LSTRA lymphoma and B16 melanoma cells was studied. Pretreatment of mice with PSK significantly increased survival after the injection of either type of sarcoma cells, although the effect was attenuated when high numbers of cells were injected. Survival was not modified significantly in LSTRA lymphoma or B16 melanoma. Mice pretreated with anti-asialo GMI serum showed significantly decreased survival from all tumors in comparison with untreated mice injected with tumors, regardless of cell dose used. We observed an inverse correlation between H-2 antigen expression and in vitro NK sensitivity of tumor cells from all lines except B16 melanoma cells. These results clearly suggest that pretreatment of mice with PSK prolongs survival and inhibits metastasis formation in mice injected with sarcoma cells, being this effect highly selective, since survival was not improved in mice injected with LSTRA lymphoma or B16 melanoma.

Protein-bound polysaccharide (PSK) induces cytotoxic activity in the NKL human natural killer cell line.

We studied the effect of protein-bound polysaccharide PSK on the activation of the human natural killer cell line NKL. We observed an increased natural killer cytotoxic activity against different tumor cells (K562, Daudi, and U937) when a standard 2- to 3-h 51chromium release assay was performed. The results parallel those obtained after treatment of the NKL cell line with interleukin-2. The highest cytotoxic activity was reached at a concentration of 100 microg/ml of PSK. This natural killer activation was inhibited when the PSK dose was 1,000 microg/ml. None of the cell surface markers that were analyzed by fluorescence-activated cell sorting showed variations after PSK or interleukin-2 treatment of NKL cells. These markers included CD2, CD11b, CD11c, CD18, CD16, CD54, CD56, CD98, CD25, CD122, HLA class I, HLA class II, CD94, ILT2, p58.1, p70, and NKp46. One of these markers (NKp46) is a major triggering receptor reported to be involved in the natural cytotoxicity of fresh or cultured human natural killer cells. In our study, another triggering receptor must be implicated in PSK-induced natural killer lysis. Our data suggest that PSK is an important biological response modifier of natural killer cells in vitro and may prove to be useful for the study of human natural killer cell biology.

Selective upregulation of MHC class I expression in metastatic colonies derived from tumor clones of a murine fibrosarcoma.

Eighteen metastatic nodes derived from the wild-type (GR9) and from 4 different clones (G2, D8, B10, and B9) obtained from a fibrosarcoma were analyzed for H-2 class I and II expression, as well as for adhesion molecules (CD44, CDIIb, CD18, CD49, and CD54). When metastatic nodes were cultured, typed for H-2 antigens, and compared with the H-2 expression of the inducing tumor cell, H-2 Kd and Dd class I expression was greater in most nodes analyzed. In contrast, the Ld molecule remained negative, or showed a minor increase. Class II expression was negative in the wild-type and the tumor clones, and remained so in the metastatic colonies. Analysis of the adhesion molecules revealed no differences between the inducing tumor cells and the metastatic nodes. The only molecule expressed was CD44, which was present in all cells studied and was also inducible by interferon-gamma. The increase in H-2K and H-2D expression was associated with resistance to natural killer cytotoxicity, as observed in the G2 tumor clone and some autologous metastases, such as B9MP2, G2MK2, and G2MLI. In three independent clones of this tumor system (D8, BIOMP6, and B9MP6) we found that tumor cells treated with interferon-gamma had the same altered phenotype, i.e., a selective lack of response of the Ld molecule to induction. These findings add a cautionary note to the well-established idea that tumor cells may lose all class I antigens during tumor progression, and suggest that sometimes this may not be the case. The selective downregulation of Ld and upregulation of Kd and Dd class I expression may give some tumor cells means of escaping both cytotoxic lymphocyte and natural killer immune surveillance.

c-K-ras overexpression is characteristic for metastases derived from a methylcholanthrene-induced fibrosarcoma.

We investigated the relationship between the activation of the c-myc and c-K-ras proto-oncogenes and the acquisition of metastatic potential in a methylcholanthrene-induced BALB/c fibrosarcoma. The murine fibrosarcoma GR9 was originally induced in BALB/c mice following exposure to the carcinogenic chemical 3-methylcholanthrene. To induce spontaneous metastasis, we used two tumor cell clones (B9 and G2) known to differ in their metastatic potential, local tumor growth, H-2 class I expression and sensitivity to natural killer (NK) cells. The metastatic nodes were obtained from the lung, liver and kidney. The results showed: (1) amplification of the c-myc proto-oncogene in original tumor clones as well as in all metastatic nodes; (2) mRNA overexpression without amplification of the K-ras proto-oncogene in the metastatic cells, regardless of their anatomical location; (3) no c-K-ras point mutations at codons 12 and 61, and (4) in general, a statistically significantly reduced in vitro sensitivity of metastatic tumor cells to NK cells as compared with the tumor clones used to induce them (p<0.05). These results therefore suggest that overexpressed c-K-ras mRNA is important during tumor progression, perhaps rendering metastatic tumor cells more resistant to lysis by NK cells.

Altered MHC class I antigens in tumors.

MHC class I antigens are lost or downregulated in invasive tumors compared with autologous normal tissues. This is observed in most of the newly induced experimental tumors analyzed if they are cloned before passaging in vivo. Similarly, this is observed in 40%-90% of human tumors using the available panel of anti-HLA class I monoclonal antibodies. In both systems the tumor populations are heterogeneous for H-2/HLA expression and composed of clones that express different amounts of MHC class I antigens. This heterogeneity may have a profound influence on tumor behavior, considering the role that MHC antigens play in T and natural killer cell-mediated responses. It is possible that the tumor escape mechanisms from T and natural killer cells select variants that express a particular MHC class I altered phenotype. We review the MHC changes detected in different experimental as well as human tumors and demonstrate the relevance of these altered H-2/HLA tumor phenotypes for implementing immunotherapeutic strategies based on T or natural killer cell-mediated responses.

Protein bound polysaccharide PSK abrogates more efficiently experimental metastases derived from H-2 negative than from H-2 positive fibrosarcoma tumor clones.

We studied the effect of protein-bound polysaccharide PSK on metastatic colonization of BALB/c mice after intravenous injections of different syngeneic murine H-2 positive and H-2 negative tumor clones. The tumor lines used were different clones from chemically induced fibrosarcomas (GR9.B9, an H-2 negative clone from GR9 tumor, and B7.1.B4, an H-2 positive clone from B7.1 tumor). These clones were selected because of their different sensitivity to NK cytotoxicity, which was related to MHC class I expression. Pretreatment of mice with PSK inhibited metastatic colonization derived from B9 H-2 negative tumor cells. In contrast, lung colonization of PSK treated mice injected with B7.1.B4 H-2 positive tumor cells was higher, and differences in the number of colonies between untreated and PSK treated mice were small. In several experiments the effect of PSK was attenuated to a greater degree when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM1 serum significantly increased (in all tumors and at different cell doses) the number of metastatic colonies in comparison with untreated mice injected with tumors, regardless of the cell dose used. These results clearly suggest that NK cell activation in vivo by the protein bound polysaccharide PSK abrogates metastasis formation in mice. Abrogation was dependent on the H-2 phenotype even when pretreatment consisted of a single dose of PSK. This effect, related to the NK sensitivity of the tumor target, can be used to predict the effect of PSK in vivo.

Effect of in vivo activation of natural killer (NK) cells by a tilorone analogue on the survival of mice injected intravenously with different experimental murine tumours.

We studied the effect of a tilorone analogue (RMI 10,874DA) and anti-asialo GM(1) serum on the survival of BALB/c and C57B1/6 mice after i.v. injections of different syngeneic murine tumour cells. Tumour lines used were different clones from chemically (GR9 wild type, GR9.B9, B7.1.B4, B7.1.B5, B7.2.38), and ultraviolet light (GRUV3)-induced sarcomas; B16 melanoma and LSTRA and YC8 lymphomas. Pretreatment of mice with tilorone inhibited metastatic colonization and increased survival significantly in all cases. In some tumour systems, the effect was attenuated when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM(1) serum significantly decreased (in all tumours and at different cell doses) survival in comparison with untreated mice injected with tumours, regardless of cell dose used. These results clearly suggest that NK cell activation in vivo by the tilorone analogue we tested prolongs survival and inhibits metastasis formation in mice, even when pretreatment consists of a single dose of the analogue.

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones.

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM1 abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2+ and H-2- clones were similarly inhibited. In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation between in vivo and in vitro results.

Generation and control of metastasis in experimental tumor systems; inhibition of experimental metastases by a tilorone analogue.

The role of the chemical compound RMI 10,874DA (3,6-bis[2-(dimethylamino)-ethoxyl]-9H-xanthene-9-one dihydrochloride) in the abrogation of the metastatic spread of tumor cells was studied. Pre-treatment of BALB/c mice with the RMI 10,874DA compound (referred to below as tilorone analogue) completely eliminated lung colonization of an H-2-negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. Other murine tumors, including H-2-positive and H-2-negative chemically induced fibrosarcoma clones and B16 melanoma, were also sensitive to the treatment; orally administered tilorone analogue given one day before the i.v. injection of tumor cells markedly inhibited lung colonization. The effect was not due to direct toxicity of tilorone analogue on tumor cells, but instead it was dependent on NK cells; this was suggested by the finding that anti-asialo GM, treatment of mice abrogated the effect of tilorone analogue. Kinetic studies of splenic NK activity in tilorone-treated mice showed a rapid boosting of NK-cell activity, the greatest stimulation occurring the day before removal of splenocytes for 51Cr-release assay against YAC-I target cells. These kinetics correlated with the inhibition of in vivo lung colonization after tilorone analogue treatment. Inhibition of experimental tumor metastasis was dose-dependent and was observed when animals were treated the day before or the day after tumor-cell injection. Furthermore, repeated treatment of mice with this tilorone analogue significantly reduced lung colonization.