Lack of IL-2 cytokine expression despite Il-2 messenger RNA transcription in tumor-infiltrating lymphocytes in primary human breast carcinoma: selective expression of early activation markers.

Tumor-infiltrating lymphocytes (TIL) are found in most human infiltrating ductal breast carcinomas. In studies of other tumors, TIL were capable of activation by IL-2, both in vitro and in vivo, to produce selective tumor cytolysis. Specific TIL-mediated tumor cytolysis in human breast tumors has recently been reported. The large numbers of TIL within human breast cancers imply that an immune response is occurring, since many of these cells express HLA class II as a late activation marker. However, the degree of early activation of the native TIL in breast tumors has not been fully investigated. Early activation markers CD69, CD43, and CD38 together with the IL-2R (CD25) and IL-2 cytokine were examined using mAbs and tissue section immunohistology. In situ hybridization was used to detect IL-2 mRNA (IL-2 mRNA) in parallel with immunohistochemical localization of IL-2. The results revealed the expression of CD69, CD43, and CD38, but markedly low CD25 (IL-2R) and IL-2 protein expression by the TIL. This strongly indicates that the TIL are an activated population of T cells that shows a deficiency in IL-2 protein and IL-2R expression despite adequate levels of IL-2 mRNA. The mechanism for apparent inhibition of IL-2 production and IL-2R expression in the presence of IL-2 mRNA is currently unclear; however, this may explain the relative anergic state of native TIL.

Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines.

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49:4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.

Vimentin rather than keratin expression in some hormone-independent breast cancer cell lines and in oncogene-transformed mammary epithelial cells.

To characterize differences in gene expression between hormone-dependent and hormone-independent mammary carcinoma, we cloned complementary DNAs of genes expressed in a hormone-independent breast carcinoma cell line that were not expressed in a hormone-dependent line. One clone, which was isolated in many copies, coded for the intermediate filament protein vimentin. A complementary DNA clone 1.8 kilobases long included the entire protein-coding region for vimentin. Vimentin was expressed by more than one-half of the hormone-independent breast carcinoma cell lines tested but not by the hormone-dependent cell lines. The cell lines which expressed vimentin expressed only low levels of cytokeratins. The correlation between vimentin expression and more advanced stages of mammary cell transformation was tested in a model system in which immortal, nontumorigenic human mammary epithelial cells or derivative lines transformed with v-ras-H or SV40 T-antigen were found not to express vimentin, whereas a derivative highly tumorigenic cell line transformed by both v-ras-H and T-antigen did express vimentin. Analysis of several other kinds of epithelial carcinoma cell lines showed only rare examples of vimentin expression.

Distinct mechanisms of c-myc and lymphokine gene expression in an antigen specific T cell clone.

Experiments were performed to examine the mechanisms regulating the expression of the c-myc gene and the IL-2 and interferon gamma (IFN-gamma) lymphokine genes in an antigen-specific murine T cell clone. IL-2 and the mitogenic lectin, concanavalin A (Con A), as well as the calcium ionophore ionomycin, in concert with phorbol ester, (PMA) enhanced c-myc gene transcription, but by distinct mechanisms as demonstrated by differential sensitivity to inhibition of protein synthesis and by transcriptional run-off assays using c-myc exon 1 and exon 2 probes. Induction of c-myc expression by IL-2, but not lectin or ionomycin plus phorbol ester, was inhibited in the presence of cycloheximide. IL-2 induced the transcription of both c-myc exons 1 and 2, whereas Con A primarily enhanced exon 1 to exon 2 transcriptional read-through. A direct relationship was observed between the level of early c-myc expression following IL-2 stimulation and the magnitude of the subsequent clonal proliferative response. Lymphokine gene expression was enhanced by Con A, but not by IL-2. Induction of the lymphokine genes in this T cell clone was under predominant post-transcriptional control and was sensitive to protein synthesis inhibition. Therefore, mitogenic lectins induce c-myc and lymphokine gene expression via different pathways.

Influence of bone marrow-derived Ia-bearing cells on the selection of the T-cell repertoire.

We have presented the results of studies using chimeric animals to examine the role of distinct cell subsets in shaping the TCR repertoire. Examination of specificity and TCR expression in antigen-specific long-term T-cell lines derived from these chimeras suggests that bone marrow-derived APC play a role in the selection of T cells for MHC restriction during development, and also profoundly influence the expansion of the TCR repertoire following antigen priming. An understanding of the molecular mechanisms that govern TCR repertoire maturation may await the development of effective in vitro model systems of T-cell differentiation.

Distinct signals are required for proliferation and lymphokine gene expression in murine T cell clones.

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.

Hybridoma 2.4G2: dissociation of the B-lymphocyte-triggering activity from the monoclonal anti-Fc IgG receptor antibody.

Previously, we have reported that monoclonal antibody specific for mouse Fc IgG receptors (Fc gamma R) [purified by affinity chromatography using goat F(ab')2 anti-rat Ig linked to Sepharose from supernatants of hybridoma 2.4G2] can induce both proliferation and antibody secretion of normal B-lymphocytes in the absence of T-lymphocytes. The current studies have shown that the B-cell-triggering activity is associated with a substance produced by 2.4G2 which can be distinguished from the 2.4G2 antibody by several criteria: (1) different preparations of 2.4G2 antibody with identical binding capacity for Fc gamma R differ markedly in their ability to trigger B-lymphocytes; (2) the B-lymphocyte-triggering substance and the anti-Fc gamma R antibody can be separated using insolubilized monoclonal mouse anti-rat kappa-antibody columns; and (3) the B-lymphocyte-triggering substance has a mol. wt of less than 50,000, while the 2.4G2 antibody has a mol. wt of 160,000. The B-lymphocyte-triggering moiety produced by the 2.4G2 hybridoma may be a previously undescribed lymphokine.

Failure of short-term treatment with flurbiprofen to enhance the therapeutic effect of cyclophosphamide against rodent sarcomas and a leukaemia.

Animals bearing metastatic fibrosarcomas were treated with cyclophosphamide (CY) alone or in combination with flurbiprofen (FP), an inhibitor of prostaglandin synthesis. FP did not affect local growth of fibrosarcomas, and the incidence of distant metastases after resection of the "primary" implants was comparable in treated and control groups. Treatment with CY retarded growth of the fibrosarcomas and reduced the proportion of animals which succumbed to metastases, but this was not altered significantly by additional treatment with FP. FP did not affect the survival of rats bearing a lymphoid leukaemia. The lifespan of animals treated with CY was increased significantly, but the concomitant administration of FP did not enhance this effect.

Effect of cyclosporin A on the growth and spontaneous metastasis of syngeneic animal tumours.

Cyclosporin A (Cy A), a novel immunosuppressive agent with apparently selective inhibitory effects on T lymphocytes and little myelotoxicity, was tested for its effects on a variety of syngeneic animal tumours including sarcomas, carcinomas and a T-cell lymphoma. Cy A, given orally or parenterally in repeated doses, had no effect on the growth rates of any of the tumours tested, but a highly significant effect on metastasis was seen in many cases. All the sarcomas examined in both rats and mice, and also the lymphoma, showed a marked increase in their metastases, in some cases even when administration of Cy A was delayed until after excision of the "primary" tumour implants. In contrast no effect of Cy A on metastasis was observed in animals bearing poorly immunogenic mammary or squamous-cell carcinomas. The metastases developing in Cy A-treated animals, when transplanted into normal syngeneic animals, showed no evidence of enhanced metastatic potential compared with their "parent tumours.