Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver

Summary The isolation and characterization of human liver cell lines are rather difficult due to limited material and poor growth in cell culture. In this report, we present the isolation, culture and characterization of a new epithelial-like liver cell line (AKN-1) with a heterogeneous cell population and many characteristics of the biliary epithelium. The AKN-1 cell line stained positively with antibodies to epithelial cytokeratin polypetides CK 8, 18, and 19. In addition, the cell line expressed the anti-human epithelial-related antigen (MOC-31), the human epithelial antigen (HEA), and the gamma-glutamyl transpeptidase, the hematopoietic growth factor, stem cell factor, and also its receptor, c-kit. The cell line failed to express albumin and factor 8 by immunohistochemistry. It did show, however, a twofold increase in 7-ethoxyresorufin-O-deethylase activity. Cytogenetic characterization revealed rare breakpoints in chromosome 2, which to our knowledge, have not yet been reported in liver cells.     

Alloreactivity against IE-encoded antigens: evidence of the discrepancy between graft rejection and reactivity of IE-reactive T cells.

Participation of IE antigens (Ag) in immune response as the transplantation Ag was examined. IE- B10.A(4R)(4R; Kk, IAk, IE-, Db) mice could not reject skin graft from IE Ag alone-disparate B10.A(2R) (2R; Kk, IAk, IEk, Db) mice despite intravenous (iv) injection of 2R spleen cells (SC) before or after skin grafting, indicating that graft rejection could not be caused across IE Ag-barrier alone. Furthermore, 4R SC could not induce lethal graft-versus-host disease (GVHD) in supralethally (950 rad) irradiated 2R mice. On the other hand, infiltration of lymphoid cells was observed at the site of transplanted 2R skin in 4R mice. SC of 4R mice unprimed or primed with 2R skin or 2R SC showed the capability to proliferate in vitro in response to 2R Ag. In immunofluorescence analysis of lymph node cells (LNC) of 4R mice injected iv with 2R SC 7 days earlier, IE-reactive CD4+Vbeta 11+ T cells did not change in number, but slightly increased the expression of interleukin-2 receptor (IL-2R). In 2R mice irradiated with 670 rad and injected iv with 4R SC 7 days earlier, 4R-derived CD4+V beta 11+ T cells proliferated, changed to blastoid form, and showed a markedly increased expression of IL-2R. To further investigate the influence of IE alloantigens on transplantation immunity, IL-2 production and anti-class I CTL activity were assayed. The 4R SC capable of recognizing IEk and Dk Ag of B10.BR (Kk, IAk, IEk, Dk) generated levels of both IL-2 and CTL activities higher than those of 2R SC capable of recognizing Dk Ag alone. These results strongly suggest that IE alloantigens indirectly act as the transplantation Ag by the stimulation of IE-reactive CD4+ helper T cells resulting in the differentiation of class I-restricted CD8+ T cells.     

Adoptive transfer of H-2-incompatible lymphokine-activated killer (LAK) cells: an approach for successful cancer immunotherapy free from graft-versus-host disease (GVHD) using murine models

We investigated whether the adoptive transfer of H-2-incompatible lymphokine-activated killer (LAK) cells would efficiently demonstrate antitumor activity without damaging the normal host cells. Allogeneic LAK cells (5 X 10(7] did not cause graft-versus-host disease (GVHD) in irradiated recipients, whereas more than half of the mice transferred with the same dose of fresh allogeneic spleen cells developed GVHD. Repeated transfer (three times at 4-day intervals, 1.2 X 10(8) cells/mouse) did not result in GVHD. Graft-versus-host reaction (GVHR), which is detectable by spleen enlargement of recipients transferred with allogeneic lymphoid cells was also absent in LAK cell-transferred mice of all strain combinations tested. Host immune responses were not affected in these mice. Therefore, it is feasible to transfer allogeneic LAK cells. With the antitumor efficacy of allogeneic LAK cells, they preferentially lysed allogeneic tumor targets. Adoptive transfer of the allogeneic LAK cells led to a significant decrease in the lung-colonizing foci of intravenously inoculated B16 melanoma cells. Allogeneic LAK cells and syngeneic ones were equally active, in vivo. The use of allogeneic LAK cells may prove to be a valuable method for effective clinical antitumor immunotherapy.     

Impairment of T cell-mediated immunity to Listeria monocytogenes in pregnant mice

In order to study pregnancy-induced changes in cell-mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant-mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria-immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti-Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.     

Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion

Induction and suppression of splenomegaly and cytotoxicity against C57BL/6 cells were studied in (AKR × C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.     

Cellular elements in the resistance to candida infection in mice. I. Contribution of T lymphocytes and phagocytes at various stages of infection.

Live organisms (cfu) of Candida albicans per organ were counted 1 hr and 1 to 20 days after an intravenous inoculation into various groups of mice which had distinct levels of immunologic or non-immunologic defense mechanisms. a) The number of cfu in the liver decreased progressively in normal mice, but those in the kidney maintained a constant level during the observation period. b) The number of cfu in the liver decreased progressively also in nude mice. In their kidneys, however, cfu increased progressively at a late stage of infection. c) In lethally irradiated AKR of nude mice in which phagocyte functions were severely depressed, the number of cfu increased progressively in both liver and kidney from the initial stage of infection. d) In immunized AKR mice, growth of C. albicans was suppressed at late stages of infection. Such protective immunity could be transferred partly with immune lymphoid cells but not with hyperimmune serum in the experimental system employed. In protection against candida infection, non-immune phagocytosis and T cell-mediated immunity appear to be required at the early and late stages of infection, respectively.