Detection of circulating tumour cells with the magnetic activated cell sorter.

A technique has been developed for the detection of tumour cells in blood with the magnetic activated cell sorter (MACS). Colonic carcinoma cell lines and disaggregated primary tumours were used to establish optimal conditions of separation. A murine monoclonal antibody specific for epithelial cells was added to the suspension of leucocytes and tumour cells, followed by magnetic labelled goat antimouse antibody. The labelled tumour cells were retrieved by passing this suspension through a MACS separation column in a strong magnetic field. Tumour cells were detected at a dilution of 10 cells per ml blood. Tumour cells were identified in mesenteric blood in three of 24 patients undergoing surgery for colorectal cancer. This study supports the use of the MACS to detect circulating tumour cells.

Lymphocytes infiltrating colorectal cancer have low proliferative capacity but can secrete normal levels of interferon gamma.

Significant numbers of infiltrating mononuclear cells are commonly observed in solid tumours, although their role in restricting tumour growth is not clear. Tumour-infiltrating lymphocytes (TIL) from 38 patients with colorectal cancer, in parallel with peripheral blood lymphocytes (PBL), were assayed to determine their ability to proliferate in response to concanavalin A (ConA), interleukin-2 (IL-2), ConA+IL-2, phorbol 12-myristate 13-acetate (PMA)+ionomycin ionomycin (IOM), and staphylococcal enterotoxin B(SEB). These reagents were selected to give a range of weak to strong proliferative responses either via or independent of the T cell receptor. Proliferation of TIL was significantly lower than that of PBL in all cultures: ConA (P < 0.001), IL-2 (P = 0.002), ConA+IL-2 (P < 0.001), PMA+IOM (P < 0.001), SEB (P = 0.002). In addition to the low proliferative capacity of TIL, production of cytokines by TIL may also play a role in control of tumour growth. We have assayed IFN gamma production in the supernatants from 16 paired TIL and PBL cultures, and tumour necrosis factor alpha (TNF alpha) in 6 paired cultures. TNF alpha concentrations were significantly lower in TIL cultures than in PBL cultures stimulated with ConA (P < 0.05), but no different in control or IL-2 stimulated cultures. IFN gamma levels did not significantly differ between PBL and TIL cultures, indicating that despite the restricted proliferative capacity of TIL, these cells remain capable of secreting significant amounts of IFN gamma.

The loss of class II MHC antigen expression by ras-transformed murine fibroblasts passaged as tumours correlates with increased tumorigenicity but is not mediated by T cells.

In several murine tumour systems, expression of class II major histocompatibility complex (MHC) antigens by tumour cells, either constitutive or inducible, correlates with reduced tumorigenicity as compared with equivalent class-II-negative cells, and CD4 phenotype T cells together with interferon gamma (which induces the expression of class II) may be involved in the control of the proliferation of class-II-expressing tumours. This implies a potential T-cell-mediated selection pressure against class II expression. To test this possibility, we have repeatedly passaged as tumours in euthymic, syngeneic mice ras-transformed murine fibroblast lines, which are class-II-inducible, to determine whether class-II-non-inducible variants are selected. We examined the expression of both class I and class II antigen in tumour cells re-established in vitro. It was found that the inducibility of class II, but not class I, expression rapidly diminished, correlating with augmented tumorigenicity. However, this loss of class II inducibility occurred in athymic as well as euthymic mice. Therefore, despite the fact that the tumorigenicity of these lines is augmented in euthymic mice depleted of CD4 T cells or interferon gamma, we found no evidence of T-cell-mediated selection against class II expression. The loss of class II expression observed must be due to mechanisms other than immune selection. The possibility that this might result from other soluble factors modulating the response to interferon gamma in vivo is discussed.

Reduced stimulation of helper T cells by Ki-ras transformed cells.

A number of viral genes and cellular oncogenes inhibit major histocompatibility complex (MHC) antigen expression at the cell surface. In the case of inhibition of class I MHC antigens by viral genes this results in a reduced recognition by antigen-specific cytotoxic T cells. The activated Ki-ras cellular oncogene carried by the Ki-murine sarcoma virus (Ki-MuSV) in contrast inhibits class II MHC (or Ia) antigen expression on transformed cells. We have studied how transformation with Ki-ras affects recognition by alloreactive helper T cells. We found that the Ki-ras inhibition of class II MHC antigen expression led to greatly reduced stimulation of alloreactive T cells to proliferate and to secrete interferon-gamma (IFN-gamma). These findings support our hypothesis that the ability of an oncogene to reduce class II MHC antigen expression is crucial to its ability to produce tumour cells.

Inducibility of class II major histocompatibility complex antigens by interferon gamma is associated with reduced tumorigenicity in C3H mouse fibroblasts transformed by v-Ki-ras.

Paired lines of C3H mouse fibroblasts transformed with murine sarcoma virus (Kirsten strain) were prepared that express high or low levels of class II major histocompatibility complex antigen after treatment with interferon gamma (IFN-gamma). Here, we described a comparison of the tumorigenicity of these lines in euthymic syngeneic and thymus-deficient nu/nu mice and in mice depleted of IFN-gamma. The class II-inducible cells are clearly less tumorigenic than the noninducible cells in syngeneic mice, but of similar tumorigenicity in nu/nu mice and in mice treated with antibodies to deplete IFN-gamma. We propose that in this system, IFN-gamma induction of class II antigens on the tumor cell surface operates to limit tumor growth; ras expression, which inhibits induction of class II antigens, prevents this and so allows tumor growth.

Instability of expression of major histocompatibility antigens in fibroblasts expressing activated ras oncogene: constitutive and interferon-gamma induced class I and class II antigens in a series of clonal isolates of murine fibroblasts transformed by v-Ki-ras.

We have examined the expression of major histocompatibility complex (MHC) antigens, constitutive or induced with interferon gamma (IFN-gamma), in a line of C3H mouse embryo fibroblasts (C3H 201) transformed with a helper-virus-free preparation of the Kirsten strain of murine sarcoma virus. C3H 201 cells expressed some class I antigen (H-2Kk) in the absence of added interferon, unlike the parental C3H 10T1/2 cells from which they were derived. However, this declined with (in vitro) passage level after transformation. Treatment with IFN-gamma induced very high expression of H-2Kk at all passage levels. There was no constitutive expression of class II antigen (I-Ak); however, this could be induced by IFN-gamma. Inducibility of I-Ak was found also to be related to the number of passages after transformation; at early passage levels after transformation more I-Ak was induced than after the cells had been allowed to grow for several passages, until at high passage levels little or no I-Ak was induced. This was not due to the presence of a subpopulation of untransformed cells since when the cells were cloned shortly after infection all the resulting clones were transformed. In addition, IFN-gamma at any passage level induced clearly less I-Ak than was found in C3H 10T1/2 cells, in which I-Ak inducibility was high and stable. Twenty-one clones were derived from C3H 201 cells at early passage (less than 8) either from soft agar or from liquid culture. These clones showed a wide variation in MHC antigen phenotype. Many expressed H-2Kk in the absence of IFN-gamma, and all were strongly inducible for H-2Kk. None showed I-Ak in the absence of IFN-gamma. All but two expressed I-Ak after IFN-gamma treatment but, with four exceptions, clearly less than the untransformed line. Four clones derived at late passage (40) resembled the late passage line. The expression of the ras oncogene and tumorigenicity was studied in representative clones; there was no obvious correlation with MHC phenotype, nor with the method of cloning. We conclude from these studies that the expression of MHC antigens by fibroblasts expressing the v-Ki-ras oncogene, either with or without exposure to interferon gamma, is unstable, varying with the number of cell generations from transformation and from clone to clone.

Interaction of v-Ki-ras oncogene and interferon-gamma in the control of histocompatibility antigen expression in mouse fibroblasts.

C3H10T1/2 fibroblasts when transformed with Kirsten murine sarcoma virus lose the ability to be induced to express class II major histocompatibility complex antigens when induced with interferon-gamma (IFN-gamma). Sublines were derived from transformed lines by cell sorting after treatment with IFN-gamma, sorting for low or high expression of H-2Ak. These sublines remained stably noninducible or inducible for class II antigen for several passages after sorting. In all other respects tested, viz, sensitivity to IFN-gamma for the generation of an antiviral state or the induction of class I antigen, content of ras gene products, the sorted sublines were very similar. We conclude that ras oncogene expression in these cells can influence the induction of class II antigen but that because ras expression in the sorted lines is similar the effect of ras expression is indirect and presumably involves interaction with other cellular factors.

T-cell immunity to murine Moloney sarcoma virus-induced tumours: L3T4+ T cells are necessary for resistance to primary sarcoma growth, but Lyt-2+ T cells are required for resistance to secondary tumour cell challenge.

Experimental protocols have been devised to deliniate the importance of T-cell subsets in immunity to Moloney sarcoma virus-induced tumours using the surface antigens L3T4 and Lyt-2 as markers of helper and cytotoxic cells, respectively. Because the monoclonal antibodies used have been shown to deplete T-cell subsets in vivo, we have been able to study the role of L3T4+ and Lyt-2+ T cells in the primary response to MSV for the first time. The results clearly show that L3T4+ T cells are the most important in resistance to the viral challenge. Mice injected with monoclonal antibodies to L3T4 grew large tumours following injection of a viral innoculum that was resisted by untreated mice or mice injected with monoclonal antibodies to Lyt-2. The same monoclonal antibodies were used to remove primed L3T4+ or Lyt-2+ T cells in vitro in adoptive transfer experiments. Normal unirradiated mice were protected from a challenge of WR19L lymphoma cells when they were given primed spleen and lymph node cells intraperitoneally. Depletion of Lyt-2+ T cells before adoptive transfer abolished this protective effect. Depletion of L3T4+ cells had no effect on the ability of primed cells to transfer immunity. Thus, while L3T4+ T cells are required for the primary rejection of MSV, only primed Lyt-2+ T cells are able to transfer resistance to a secondary challenge of lymphoma cells.

The in vivo fate of a 211At labelled monoclonal antibody with known specificity in a murine system.

A monoclonal antibody reactive against the human transferrin receptor has been labelled with the alpha and X ray emitting isotope Astatine 211. The labelling procedure does not affect the ability of the product to bind to the transferrin receptor on the human leukemic cell line HL60. Using a direct binding assay, 211At labelled antibody can be specifically inhibited from binding to its target cells by excess unlabelled antibody. Furthermore, the binding inhibition demonstrated in this system correlates to enhanced clonogenic survival of these cells, indicating that very few atoms of 211At/cell are required for cell death. Data obtained from labelled antibody injected into mice show that the labelled product in serum retains the ability to bind to HL60 cells in vitro, although tissue distributions of the injected activity implies that some of the radiolabel is lost from the protein. Despite this loss of label, preliminary experiments on the localization of labelled antibody to HL60 cells growing s/c in nude mice show that tumor tissue has a higher specific activity than all other tissues, other than blood, after 12 hours. This suggests that further work on the nature of label degradation in vivo is warranted in the context of potential therapeutic and diagnostic studies.

The specific inhibition of cellular clonogenic proliferation using 211At labelled lectins and antibodies--I.

A monoclonal antibody with a broad anti-human leucocyte specificity, designated BK 19.45 and the plant lectin Concanavalin A have been labelled with the alpha-emitting cyclotron produced radiohalogen astatine-211. Both human and murine tumour cell lines and human leukemic bone marrow samples have been specifically labelled with these radioactive proteins. In all cases the amount of 211At bound to the cells is directly correlated with a decrease in cellular reproductive potential as shown by the ability of the cells to proliferate in a clonogenic assay. For the labelled monoclonal antibody experiments, the radiation dose needed to yield 37% cell survival, the D37 dose, may be achieved with an average of 12 211At atoms/cell.