Antiestrogen potentiation of antiproliferative effects of vitamin D3 analogues in breast cancer cells.

[3H]thymidine incorporation and DNA content were used to investigate the antiproliferative effects of 1,25(OH)2D3 and four analogues [16-ene-1,25(OH)2D3 (16-ene)]; 16-ene,23-yne-1,25(0H)2,D3; EB1089; and 22 oxa-1,25(OH)2D3] on MCF-7, BT-474, and MDA-MB-453 breast cancer cell lines. 1,25(OH)2D3 and the analogues elicited a biphasic response from MCF-7 and BT-474 estrogen receptor (ER)-positive cells, in the presence of estradiol (E2), with lower doses (between 10(-12) and 10(-10) M) tending to stimulate proliferation and higher doses (between 10(-9) and 10(-6) M) inhibiting proliferation by as much as 65%. In the absence of E2, the stimulatory effect was abrogated. Proliferation of MDA-MB-453, estrogen receptor-negative (ER-) cells, was stimulated by these compounds only at 10(-12) M, and inhibited by all higher doses, by as much as 83%. All three cell lines were shown to be vitamin D receptor (VDR) positive, and 1,25(OH)2D3 and all four analogues bound to the VDR with high affinities in each cell line. The antiestrogen ICI 164,384 inhibited the proliferation of all three cell lines. ICI 164,384 at 10(-8) M in combination with 1,25(OH)2D, or EB1089 converted biphasic response of the ER+ cells to one resembling the response of the ER- cells, by eliminating the stimulatory response elicited by 1,25(OH)2D3 at low doses and enhancing the antiproliferative effects of higher doses by as much as 1000-fold. These data are consistent with the hypothesis that E2 in the ER+ cells blocks the antiproliferative effects of the analogues and suggest the potential usefulness of combined antiestrogen and 1,25(OH)2D3 analogues in ER+ breast tumors, whereas 1,25(OH)2D3 analogues alone might suffice in ER- breast tumors.

Dendritic epidermal T cells inhibit T cell proliferation and may induce tolerance by cytotoxicity.

Hapten-conjugated cells of the dendritic epidermal T cell (DETC) line AU16 induce specific immunologic tolerance in vivo and inhibit the proliferation of native T cells in response to hapten-bearing dendritic cells in vitro. The purpose of this study was to test the hypothesis that these activities of DETC are mediated by a cytotoxic mechanism. In addition, because AU16 cells, unlike most DETC, express a TCR-alpha beta, we compared their activity in the in vivo and in vitro assays with a TCR(+)-gamma delta DETC line (T245) and an TCR(+)-alpha beta, FITC-specific T cell line (T4/28). Hapten-conjugated AU16 and T245 cells, but not T4/28 cells, induced tolerance to FITC in vivo. In addition, the proliferative response of naive T lymphocytes to gamma-irradiated, hapten-bearing APCs was inhibited by the addition of gamma-irradiated, FITC-conjugated DETC to the cultures on day 0. To determine whether the T cell or the APC was the target, AU16 cells were added to the cultures on day 4, when few APCs remained. FITC-conjugated AU16 and T245 cells profoundly inhibited [3H]thymidine incorporation by the T cells, indicating that the T cell was the target of the DETC. Furthermore, AU16 cells reduced the number of T cells in the in vitro proliferation assay, suggesting that inhibition of [3H]thymidine incorporation was a result of the cytotoxic activity of DETC. We speculate that cytotoxicity may also account for the ability of DETC to induce tolerance in vivo.

Inhibitory effect of a dendritic epidermal T cell line on K1735 melanoma cells in vivo and in vitro.

Murine epidermis contains a resident population of dendritic epidermal T cells (DETCs) with unknown function. We tested the ability of a cultured line of these cells to inhibit the growth of murine melanoma cells in vitro and in vivo. The interleukin-2 (IL-2)-dependent T cell line AU16, derived from the epidermis of C3H mice, was used as the source of DETCs. AU16 cells were cytotoxic to syngeneic K1735 and CM3205 C3H melanoma cell lines, as well as to a chemically induced cutaneous fibrosarcoma, in an in vitro 51Cr release assay. Mixing viable AU16 cells, but not splenic T cells, with K1735 melanoma cells immediately before intradermal injection into the flank of mice prevented or delayed the outgrowth of melanomas. AU16 cells killed by freezing and thawing and supernatants from cultures of AU16 cells did not inhibit melanoma growth in vivo or in vitro. Injection of viable AU16 cells intravenously or at a separate intradermal site had no effect on melanoma growth. These studies suggest that IL-2-activated DETCs have the potential to inhibit the growth of melanoma cells in vivo and that their cytotoxic activity may be responsible for this effect.

Studies on the mechanism of immunologic tolerance induction by murine dendritic epidermal Thy-1+ cell lines.

We are investigating the functions of the Thy-1+ dendritic cells present in murine epidermis. Dendritic epidermal Thy-1+ cell (DETC) lines conjugated in vitro with hapten induce specific immunologic tolerance upon intravenous (i.v.) or subcutaneous injection into the footpad of normal mice. In these studies, we demonstrate that hapten-conjugated cells of other long-term, interleukin-2 (IL-2)-dependent, T cell lines are unable to induce tolerance upon footpad injection, indicating that the ability of DETC lines to induce tolerance is not a function of long-term cell culture or IL-2 dependence. Suppressor T cells were not found in mice made tolerant by footpad injection of hapten-conjugated cells of the DETC line AU16, although they could be demonstrated in mice made tolerant by i.v. injection. Studies of lymphocyte proliferation in vitro suggested that hapten-conjugated AU16 cells may induce tolerance by inhibiting the proliferation of activated T lymphocytes.

F(ab')2 anti-CD4 and intact anti-CD4 monoclonal antibodies inhibit the accumulation of CD4+ T cells, CD8+ T cells, and B cells in the kidneys of lupus-prone NZB/NZW mice.

Murine lupus in NZB/NZW (B/W) mice is characterized by immune-complex glomerulonephritis and lymphocytic infiltration of several organs, including the kidney. We recently showed that treatment of B/W mice with F(ab')2 anti-CD4 monoclonal antibody retards autoimmunity by inhibiting the function of CD4+ cells and not by depleting them. To determine if treatment with F(ab')2 anti-CD4 prevented lymphocytic infiltration of kidneys or simply inhibited the function of the infiltrating lymphocytes, long-term survivors of treatment with F(ab')2 anti-CD4 and intact anti-CD4 were sacrificed for immunohistochemical analysis of their kidneys. Untreated B/W mice had large lymphocytic aggregates under the surface epithelium of the renal calyces. Most of these lymphocytes were CD4+ T cells, but CD8+ T cells and B cells were also present. In contrast, treatment with either intact anti-CD4 or F(ab')2 anti-CD4 substantially reduced, and in many cases prevented, the development of renal infiltrates. Treatment with either form of anti-CD4 not only inhibited renal infiltration by CD4+ T cells, but also prevented the accumulation of CD8+ T cells and B cells. These observations suggest a role for the CD4+ T cell in the accumulation of lymphocytes in target organs.

Treatment of murine lupus with F(ab')2 fragments of monoclonal antibody to L3T4. Suppression of autoimmunity does not depend on T helper cell depletion.

Treatment with mAb to the L3T4 Ag on Th cells can inhibit autoimmunity in mice. However, the mechanism by which anti-L3T4 inhibits autoimmunity is not known. In these studies, lupus-prone NZB/NZW F1 (B/W) mice were treated with F(ab')2 fragments of mAb to L3T4 to determine whether Th cell depletion is required for the beneficial effects of anti-L3T4. We first showed that treatment of female B/W mice with F(ab')2 anti-L3T4 from age 5 to 9 mo significantly reduced autoantibody production without depleting L3T4+ cells. However, treatment was complicated by the development of a host immune response to the rat mAb fragments. To circumvent this problem, female B/W mice were treated with a single high-dose of intact rat mAb to L3T4 (GK1.5) at age two mo. to induce immune tolerance to the mAb. Then, after recovery of L3T4+ cells, the mice were treated from age four to 14 mo with either F(ab')2 anti-L3T4 (0.5 mg 3 times per wk), intact anti-L3T4, or saline. In mice tolerized by this regimen, neither the F(ab')2 rat mAb nor the intact rat mAb elicited a host response. The mAb fragments bound target Ag but did not deplete the Th cells, whereas intact mAb to L3T4 profoundly depleted the L3T4+ cells. Despite this difference, both therapies had the same substantial beneficial effects on autoimmunity. They significantly decreased anti-DNA Ab production, improved renal function and prolonged survival. The initial tolerizing dose, by itself, did not inhibit autoimmunity. These findings show that anti-L3T4 suppresses autoimmunity by directly altering Th cell function through the L3T4 Ag, and not solely by depleting Th cells. They also document the detrimental effects of the host immune response to therapy with anti-L3T4 mAb, and they demonstrate a new strategy by which this response may be prevented.