The role of receptor-ligand endocytosis and degradation in interleukin-2 signaling and T-lymphocyte proliferation.

The specific intracellular signaling pathways for interleukin-2 (IL-2) that lead to delivery of the proliferative stimulus are currently unknown. We and others have excluded signaling pathways used by other growth factors and by the antigen-specific T-cell receptor, such as increased intracellular Ca2+ concentrations, activation of protein kinase C, or ion transport across the plasma membrane. One feature of IL-2 signaling that may be important in delivery of the proliferative stimulus is endocytosis and processing of the lymphokine receptor-ligand complex. In this study we examined these steps in receptor signaling by mouse CTLL-2 cells and human OKT3-activated T-cells using monoclonal antibodies specific for the 55 kDa alpha-subunit of the IL-2R that allow IL-2 binding but block endocytosis, and with lysosomotrophic amines that selectively inhibit receptor mediated endocytosis and/or processing of IL-2. Our results demonstrate that these inhibitors block receptor endocytosis, ligand degradation, c-fos protooncogene activation, and ultimately proliferation of the IL-2-dependent T-cell line, CTLL-2. In heterogeneous populations of activated human T cells the lysosomotrophic amines demonstrated a greater inhibition of degradation than of endocytosis. These observations support the hypothesis that IL-2/IL-2R endocytosis and ligand/receptor processing or degradation may be important steps in lymphokine signal transduction.

Characterization of variant and parental-cross-protective immunity to immunogenic variants of a murine fibrosarcoma using the local adoptive transfer assay.

The purpose of this study was to characterize the lymphocyte populations responsible for rejection of immunogenic (Imm+) tumor variants, and the cross-protective immunity engendered by Imm+ variants against the weakly immunogenic parental tumor. Immunogenic clones of the weakly immunogenic methylcholanthrene-induced fibrosarcoma MCA-F have been generated using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2'-deoxycytidine, or ultraviolet radiation (UV-B; 280-320 nm). These clones grow progressively in immunosuppressed adult-thymectomized irradiated mice, but are rejected by immunocompetent syngeneic hosts. The parental MCA-F tumor grows progressively in both groups. Mice that have rejected a challenge of 1 x 10(5) Imm+ cells show an anamnestic immune response against both the Imm+ clone and the parental MCA-F tumor. Using the local adoptive transfer assay and depletion of T-cell subsets with antibody plus complement, we show that immunity induced by the Imm+ variants against the parent MCA-F was mediated by the Thy1.2+, L3T4a+ population without an apparent contribution by Lyt2.1+ cells. Although antivariant immunity was also dependent upon Thy1.2+ cells, depletion of either the L3T4a+ or the Lyt2.1+ cells failed to abolish immunity against the variant. A role for Lyt2.1+ T lymphocytes in antivariant immunity, but not antiparent immunity, was supported by the results of cytotoxic T lymphocyte (CTL) assays. Following immunization with high numbers (1 x 10(5) to 5 x 10(5) of viable Imm+ cells, antivariant, but not antiparent CTL activity was detected in mixed lymphocyte tumor cell cultures. Immunization with lower numbers (3 x 10(4] of viable Imm+ or with high numbers of mitomycin-C-treated Imm+ engenders only antivariant immunity without parental cross-protection. Under these conditions lymphocytes mediating immunity against the variant in the local adoptive transfer assay were exclusively of the Thy1.2+, L3T4a+ phenotype, with no contribution from the Lyt2.1+ cells. Identical results were obtained for Imm+ clones of MCA-F induced by methylnitronitrosoguanidine, 5-azadeoxycytidine, and UV-B, suggesting that the nature of the antitumor immunity engendered by Imm+ is not significantly affected by the agent used. Furthermore, these results demonstrate that the cross-reactivity and cellular effectors of antitumor immunity in this system are influenced by the immunizing dose of Imm+ cells: the predominant effectors of both antivariant and parental-cross-reactive immunity were of the CD4+ T cell subclass, with a CD8+ cytotoxic population contributing to antivariant immunity only after high-dose immunization.

Differential extraction of tumor-specific antigens from two ultraviolet light-induced murine fibrosarcomas with the use of 1-butanol.

The purpose of this study was to investigate the immunobiological characteristics of the tumor-specific cell surface antigen expressed by the UV-induced murine fibrosarcoma, UV-2240. UV-2240 is classified as a regressor UV tumor because it is immunologically rejected by normal syngeneic mice but grows in immunocompromised or UV-irradiated hosts. The strong tumor-specific rejection antigen expressed by UV-2240 was found on the plasma membrane, and unlike the previously characterized antigen of UV-1591, the UV-2240 antigen was removed by using the noncytolytic butanol extraction technique. The tumor antigen activity in butanol extracts was resistant to digestion by endoglycosidase F and alpha-mannosidase, but was destroyed by pronase. In addition, the immunoprotective activity in extracts of UV-2240 was thermostable. These data demonstrate that the UV-2240-specific tumor antigen possesses physicochemical properties distinct from those of its well-characterized counterpart UV-1591.

Inhibition of T-lymphocyte proliferation by the cyclic polypeptide didemnin B: no inhibition of lymphokine stimulation.

We investigated possible mechanisms by which the cyclic depsipeptide didemnin B (DB) inhibits lymphocyte proliferation. DB inhibited the proliferation of Con-A stimulated murine splenocytes, the interleukin-2 (IL-2) dependent proliferation of the CTLL-2 cell line, and the interleukin 4 (IL 4) dependent growth of both CTLL-2 and D10.G.4.1 cell lines at approximately equimolar concentrations (SD50 = 3 to 10 X 10(-9)M). Inhibition of CTLL-2 growth by 10(-8)M DB was partially reversible, and significantly blocked the incorporation of [3H]-thymidine even when added 24 hr after initial IL 2 stimulation. Concentrations of DB (10(-8)M) that completely blocked mitogen-driven spleen cell blastogenesis only partially inhibited the synthesis and secretion of IL-2. Although DB blocked the growth of T lymphocyte clones in response to both recombinant human IL 2 and recombinant murine IL 4, the suppression was not due to an uncoupling of lymphokinetic signaling but was closely correlated with an inhibition of protein and RNA synthesis. Addition of DB to an in vitro translation system did not inhibit protein synthesis. Thus, we conclude that DB functions as an antiproliferative, and not as a specifically immunosuppressive, compound.