In vitro induction of cell-mediated immunity to murine leukemia cells

Summary An adoptive chemoimmunotherapeutic model based on the use of chemotherapy and lymphocytes specifically sensitized against tumor cells in vitro was tested in mice transplanted with syngeneic leukemia cells. C57BL/6 and A strain mice were inoculated i.p. or i.v. (day 0) with lethal doses (1×10³–1×10⁵) of EL4 and YAC leukemia cells, respectively. Leukemic mice were subsequently treated (day 1 or day 3) with partially curative doses (80–140 mg/kg) of cyclophosphamide (Cy), followed by i.p. or i.v. administration of 1–3×10⁷ cytotoxic lymphocytes (CL) induced in macro-mixed leukocyte-tumor cell cultures (MLTC). The following results were obtained: untreated mice died with tumor within 20 days; mice receiving sensitized lymphocytes only showed a modest prolongation of survival and only 5–15% of the animals were cured; treatment with Cy alone or with Cy and normal lymphocytes prolonged survival considerably and cured 20–60% of the mice; mice subjected to Cy in conjunction with in vitro-sensitized lymphoid cells, either syngeneic or allogeneic, had survival rates of 80–100% (100 days). Under the conditions employed, no severe manifestations of clinical graft-versus-host (GVH) reaction were observed. These findings imply that in vitro-sensitized immunocytes and cytoreductive drugs can operate cumulatively.     

In vivo and in vitro evaluation of cell-mediated immunity in patients with paracoccidioidomycosis

The cellular immune response to specific and nonspecific agents was investigated. both in vivo and in vitro, in 19 patients with paracoceidioidomycosis. In addition, the immunologic study of an investigator aceidentally inoculated with P. brasiliensis was included in this study. Nearly half of the patients showed depressed cell-mediated immune responses, as evaluated by intradermal tests with an antigenic preparation from P. brasiliensis (P.b.Ag.), ubiquitous antigens, and by the ability to develop sensitization to 2,4-dinitrochlorobenzene. A similar proportion of impaired responses was observed when the patients' lymphocytes were cultured with phytohemagglutinin (PHA). C'. albicans antigen and P.h.Ag. A factor was detected in the plasma of some patients which reduced the ability of patients' and normal lymphocytes to undergo blastic transformation. A positive correlation was found between the ability to develop delayed cutaneous hypersensitivity reactions to P.b.Ag. and other ubiquitous antigens, normal in vitro responsiveness to PHA and the absence of humoral blastogenic inhibitory factor. The inhibition of leukocyte migration, but not lymphocyte transformation, correlated positively with delayed hypersensitivity. The percentage of T lymphocytes was significantly reduced in the group of patients, being the absolute number and percentage of B cells bearing receptors tor complement normal. Two polar immunological patterns emerged. One characterized by positiveness in the skin test to P.b.Ag. and lack of significant abnormalities in cellular immunity, and another anergic to P.b.Ag., with cell mediated immunity severely depressed. Between the two polar groups, there were patients with intermediary patterns of immune response. This paper also includes the results obtained with the administration of transfer factor and levamisole to some of the patients.     

In vivo protection against syngeneic Gross virus-induced lymphoma in rats: comparison with in vitro studies of cell-mediated immunity.

Spleen cells at various times after inoculation of W/Fu rats with a syngeneic Gross virus-induced lymphoma, (C58NT)D, were tested for their in vivo activity in adoptive transfer experiments and for their in vitro reactivity in a 4-hr 51Cr release cytotoxicity assay and in a mixed lymphocyte-tumor cell interaction assay. In adoptive transfer, the best protection against tumor growth was observed with immune spleen cells taken at 30 days after tumor cell inoculation (the peak of reactivity in the mixed lymphocyte-tumor cell interaction assay) whereas cells taken at 10 days (the peak reactivity in the 51Cr release cytotoxicity assay) gave only partial protection. The protection detected in the adoptive transfer experiments was specific for (C58NT)D associated antigens, and this correlated well with the specificity observed in the in vitro cell-mediated immunity assays. T cells, but not complement receptor-bearing cells or macrophages, were essential for the protection against tumor growth in vivo, and also for the in vitro reactivity in the 51Cr release cytotoxicity and the mixed lymphocyte-tumor cell interaction assays.     

Clinical studies on cell-mediated immunity in patients with malignant disease

Summary In vitro mitogenic responses of lymphocytes of patients with advanced cancer of the stomach or lung were determined and the cells involved in the depressed responses of patients characterized. Proliferative responses to phytohemagglutinin and concanavalin A of lymphocyte rich mononuclear cells of the patients were impaired, but increased after in vitro unstimulated culture for seven days. Mitogenic responses were enhanced by depletion of monocytes using a Sephadex G-10 column, and further enhanced by removal of nylon wool nonadherent cells. Nylon wool nonadherent cells of the patients suppressed the mitogenic responses of autologous and allogeneic lymphocytes, but lost the suppressive activity during seven days' in vitro culture. Nonadherent cells of normal donors did not inhibit mitogenic responses. The results suggest that peripheral blood mononuclear cells of cancer patients may contain at least two populations of suppressor cells for mitogenic responses, monocytes and nylon wool nonadherent cells, which could be one of the causes of impaired mitogenic responses in these cancer patients.     

In vitro studies on information transfer in cells from allotype-suppressed rabbits.

Data are presented which show that cells from allotypically suppressed rabbits resist in vitro "rescue" attempts in which informational ribonucleic acid (i-RNA) coding for the suppressed allotypic marker is used. It is shown that peritoneal exudate cells from such thoroughly suppressed animals contain cells that yield i-RNA coding for the suppressed marker, and that central lymphatic tissue possesses cells that produce Ig of the suppressed type.     

In vitro studies on cell-mediated cytotoxicity by means of a terminal labeling technique.

A modification is described of the Takasugi-Klein assay for cell-mediated cytotoxicity, based on the measurement of ⁵¹Cr uptake by viable cells at the conclusion of effector-target cell interactions. Findings showing the applicability of this method to the quantitative determination of cell-mediated cytotoxicity against syngeneic solid tumors of mice are presented. It was found that repeated washing of splenocytes from donors with large tumors often elevated appreciably the cytotoxic capacity of the effector cells, and that washing of splenocytes from normal animals reduced their apparently nonspecific toxic effects.     

The effect of adding a lipid tail to tyrosine-azobenzenearsonate: In vivo and in vitro consequences for cell-mediated immunity

Coupling lipid tails of increasing length to the tyrosine-azobenzene-arsonate molecule resulted in a decrease in in vivo immunogenicity. When used to induce in vitro stimulation of sensitized lymphocytes a decrease in dose was required to produce an optimal response as the lipid sidechain increased in size. This was attributable to the cytotoxic effects produced by the detergent-like activity of the large lipid side-chain derivatives. When used for pulsing of macrophages the stickiness of the larger lipid derivatives made them more effective at low doses and times for initiation of lymphocyte stimulation.     

Effects of kinins, kallikrein and its inhibitor on certain indices of cell-mediated immunity and humoral immunity in rabbits

The experiments were carried out on 54 rabbits in 9 groups of 6 animals each: group I -- controls, groups II, III, IV -- bradykinin i.v., groups V, VI, VII -- Depot-Kallikrein i.m. every other day for 3 weeks, groups VIII and IX -- Traskolan (trasylol) i.v. four times at intervals of 1 or 12 hours. The determined indices of cell-mediated and humoral immunity included: phagocytic activity of the reticuloendothelial system and peripheral blood leucocytes and their leukergic adhesiveness, haemagglutinin and haemolysin levels, serum complement titre, and the number of cells forming rosettes (RFC) or plaques (PFC) in the blood and spleen. These indices were determined 15 minutes, 3 and 24 hours after bradykinin administration, after 1, 2 and 3 weeks of kallikrein administration, and 1 or 12 hours after the last dose of Traskolan. Most determined indices showed always some fall. Only the phagocytic activity of the reticuloendothelial system was moderately increased in all groups, and in the bradykinin group leucocyte phagocytosis was increased slightly while their leukergic reaction was increased very strongly.     

The effect of purified alpha-fetoprotein on in vitro assays of cell-mediated immunity.

We have evaluated the effect of purified human α-fetoprotein (AFP) on several in vitro correlates of cell-mediated immunity. AFP (100 μg/ml) had no effect on antigen-induced migration inhibitory factor (MIF) production or on the ability of T cells to bind sheep erythrocytes. In contrast, AFP had a twofold effect on mitogen-and antigen-induced lymphocyte proliferation. In a dose-dependent fashion (1–100 μg/ml), AFP was mitogenic to lymphocyte cultures and also suppressed tritiated thymidine incorporation by PHA- and SK-SD-stimulated cultures. Albumin had somewhat similar effects on lymphocyte proliferation but only at 100 μg/ml. The reason for the latter was not clear since the albumin preparation was free of any detectable AFP. Our studies suggest that human AFP may not have any biologically significant immunosuppressive function.     

Immune interferon produced in vitro as a quantitative indicator of cell-mediated immunity.

The present study was constructed to provide some information on the possibility of utilizing immune interferon as a quantitative indicator of cell-mediated immunity and to clarify some of the nature of immune interferon-producing cells (IIPC). When spleen cells derived from L cell-sensitized mice were co-cultivated with L cells, interferon appeared in the culture fluid. It was shown by additional experiments that the cells responsible for immune interferon production in this system were T-lymphocytes assisted by macrophages. The pattern of kinetics of immune interferon production in vitro was similar to that of migration inhibitory factor or T cell-mediated cytotoxicity.