B7-1, IFN gamma and anti-CTLA-4 co-operate to prevent T-cell tolerization during immunotherapy against a murine T-lymphoma.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.

Interleukin-12-activated natural killer cells recognize B7 costimulatory molecules on tumor cells and autologous dendritic cells.

Activation of natural killer (NK) cells in the presence of interleukin-12 (IL-12) augments the capacity of these effector cells to recognize B7-1- and B7-2-expressing target cells. These effector cells also efficiently lyse autologous B7-positive progenitor or organ-derived dendritic cells, suggesting a physiologic regulatory pathway between IL-12, NK cells, and B7-expressing antigen-presenting cells. Although IL-12-activated NK cells secreted higher levels of interferon-gamma, this cytokine did not play a role in synergistic effects of IL-12 and B7 on NK activation. The B7-counterreceptor was found to be selectively upregulated on IL-2/IL-12 as compared with IL-2-activated NK cells. CD28 is functionally involved in the recognition of B7 on target cells since IL-2/IL-12-activated NK cells derived from CD28 knockout mice were strongly reduced in their capacity to lyse syngeneic B7-positive tumor cells as well as antigen-presenting cells. However, recognition of B7 on allogeneic targets did not require the expression of CD28 on the IL-2/IL-12-activated NK cells. Hence, IL-12 triggers the expression of both CD28-dependent and CD28-independent mechanisms that allow NK cells to eliminate B7-positive target cells including autologous dendritic cells.

Multiple effects of transfection with interleukin 2 and/or interferon gamma on the behavior of mouse T lymphoma cells.

We have previously reported that transfection of mouse interferon gamma (IFN-gamma) in H-2Kk-positive BW variants (BW-Sp3) abolishes tumorigenicity and reduces metastatic capacity. To further increase the immunogenicity of BW-Sp3 cells, the gene for human interleukin 2 (huIL-2) was transfected in these cancer cells. Single BW-Sp3(huIL-2) and double BW-Sp3(huIL-2+IFN-gamma) transfectants were generated and their behavior was investigated as compared to parental and IFN-gamma-transfected BW-Sp3. Although expression of huIL-2 was equally effective as IFN-gamma in preventing tumor formation and reducing experimental metastasis, it did not confer protection to spontaneous metastases and even reversed the anti-metastatic activity of IFN-gamma. Inoculation of the BW variants in immunocompromised mice revealed that expression of IL-2 activates both T cells and aspecific immune effectors. However, in immunocompromised mice a clear pro-metastatic effect of IL-2 was recorded. Analysis of membrane antigens on the different variants showed a selective effect of huIL-2 on the expression of two surface antigens, i.e. L-selectin and metastatic T cell hybridoma antigen (MTH), which may contribute to metastasis. Hence upon expression of huIL-2 in T lymphoma variants, tumor outcome will depend on the balanced effects of the transfected cytokines on the immune response and the redirected effect on tumor progression.

Introduction of the interferon gamma gene into mouse T lymphoma cells with low MHC class I-expression results in selective induction of H-2Dk and concomitant enhanced metastasis.

Interferon-gamma (IFN gamma)-induced up-regulation of MHC class I expression on tumor cells can induce a potent CD8-mediated antitumor response. Consequently, many investigators have proposed IFN gamma gene transfection as a means to immunogenize tumor cells and to vaccinate against metastatic disease. In this study, we demonstrate that transfection of the IFN gamma gene in a BW5147 variant (LiDlo) with low MHC class I expression results in a selective induction of H-2Dk but unaltered H-2Kk expression. In earlier reports we demonstrated a positive correlation between H-2Dk expression and enhanced metastatic potential of BW variants. In accordance with these observations, we observed that intravenous inoculation of LiDlo(IFN gamma) variants into syngeneic AKR mice led to enhanced metastasis as compared to parental LiDlo and LiDlo(neo) control transfectants. Tumor cells, derived from local subcutaneous tumors or sporadic metastases from mice inoculated with LiDlo tumor cells, were found to up-regulate H-2Dk selectively. Anti-asialoGM1 treatment of AKR mice allowed rapid experimental metastasis formation by the LiDlo and LiDlo(neo) variants, indicating that natural killer (NK) cells control the metastatic behavior of these tumor cells. This was corroborated by in vitro cytotoxicity experiments, demonstrating the LiDlo and LiDlo(neo) tumor cells were NK-sensitive, while the BW IFN gamma transfectants became resistant to lymphokine-activated killer cells and poly(I).poly(C)-induced NK cells. We thus conclude that (a) IFN gamma up-regulates selectively the MHC class I antigen H-2Dk, (b) H-2Dk governs susceptibility towards NK cells, and (c) NK susceptibility determines the experimental metastatic behavior of BW tumor cells.

Expression of B7-1 by highly metastatic mouse T lymphomas induces optimal natural killer cell-mediated cytotoxicity.

The interaction between B7-1 and CD28 provides costimulatory signals not only for T cells but also for natural killer (NK) cells. Highly metastatic mouse T lymphoma cells (BW-Li) can escape from NK cell-mediated killing by expressing H-2Dk molecules that negatively regulate NK lytic activity. We have analyzed whether B7-1:CD28 overrules the MHC class I-mediated inactivation of NK cells by transfecting BW-Li with the gene coding for B7-1. Expression of B7-1 rendered BW-Li cells sensitive toward NK cells. The experimental metastatic capacity of the B7-1 transfectants was drastically reduced in both syngeneic AKR and SCID mice but could be restored in SCID-bg mice. These results provide direct evidence that B7-1 expression leads to NK-mediated elimination of metastasizing, NK-resistant tumor cells.