Michael A. Palladino

Effect of calorie restriction on the production and responsiveness to interleukin 2 in (NZB × NZW)F1 mice

Abstract Calorie restriction preserves the immunologic functions of the autoimmune prone B/W mice and prolongs their life. B/W mice fed normal calories were deficient in production of Interleukin 2 (IL-2) and in responses to exogenous IL-2 after 5 months of age. Calorie restriction leads to the preservation of IL-2 production in the spleen cells of these animals. Furthermore, these calorie-restricted mice responded vigorously to exogenous IL-2 in the thymocyte proliferation assay while mice fed a normal calorie diet lost much of the ability of their thymocytes to respond to IL-2. Whether calorie restriction in preserving the immune function of B/W mice involves IL-2 production and/or IL-2 response requires further analysis.

In vivo Localization of cloned IL-2-Dependent T cells

The quantitative organ distribution and tissue microenvironment positioning of radioisotopically labeled cloned T cells were characterized. Intravenous (iv) injection of 51chromium (51Cr)-labeled, long-term cultured cloned T-helper cells and cells from several cloned cytolytic T-lymphocyte lines (CTLL) resulted in poor localization of these cells in recipient lymphoid tissues, similar to results reported for activated lymphoblastoid cells. Simultaneous administration of interleukin 2 (IL-2) with labeled cells resulted in enhanced recovery from recipient spleen. By the intraperitoneal (ip) injection route, overall percentage recovery of injected radioactivity was lower than by the iv route, but significant localization to lymph nodes occurred. Examination of autoradiographs of tissue sections from recipients of [3H]adenosine-labeled cells showed most label associated with intact, isolated cells in the liver, lungs, spleen, and small intestine. By 24 hr after iv injection, labeled cells in spleen sections were distributed to both nonlymphoid and T- and B-lymphoid areas. These findings suggest that poor localization of these cells to recipient lymphoid tissue is due both to intrinsic characteristics of cultured lymphocytes and to the possible reduced viability of IL-2-dependent cells in vivo.

Different forms of Ly-5 within the T-cell lineage

The Ly-5 system, defined both by alloantibodies (Komuro et al. 1975) and by xenoantibodies (Omary et al. 1980, Siadak and Nowinski 1980), is noted for molecular differences that distinguish various hematopoietic cell lineages (Michaelson et al. 1979). A 200K (Mr 200000) is expressed by T cells, a 205K form occurs on macrophages, and a 220K form [which can be separately identified by xenoantibody (Coffman and Weissman 1981, Dalchau and Fabre 1981, Kincade et al. 1981) and may therefore identify an epitope lacking in the smaller forms] is characteristic of B cells. This relation of molecular form to cell lineage has been confirmed by ascertaining the Ly-5 molecular phenotypes of cloned cell lines representing T, B, and macrophage lineages (Tung et al. 1981). Extending our survey to a further range of cloned T-cell lines, we now find that the T lineage itself is diverse in expression of distinguishable Ly-5 molecular forms. Each of the 10 cell lines, shown in Table 1, seven of which have the phenotype Ly-23 and three the phenotype Ly-1, was examined in the usual way by cell surface radioiodination, immunoprecipitation (with monoclonal Ly-5.1 alloantibody; Shen 1981), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Figure 1 shows that all three Ly-1 lines express the familiar 200K form previously found to typify T cells, whereas all seven Ly-23 lines expressed two forms, 210K and 215K, neither of which has previously been seen in any lineage. These two newly identified forms bring the number of distinguishable Ly-5 forms to five, and they are shown in Figure 2 to be separable from the three other previously known forms. All five forms were recognizable in combined lysates of spleen cells and CTLL-R8 Ly-23 cells (Fig. 2). The reason why the 210K and 215K forms were not observed before is probably that Ly-23 is a small cell set that requires cloning or other enrichment to provide enough material.

Characterization of interleukin 2 (IL-2)-dependent cytotoxic T-cell clones. V. Transfer of resistance to allografts and tumor grafts requires exogenous IL-2.

The adoptive transfer of resistance to tumor grafts with cloned interleukin 2 (IL-2)-dependent cytotoxic T-cell lines was examined. Two clones were used: clone CTLL-A2 which recognizes H-2Dd determinants and clone CTLL-R5 which recognizes a unique cell surface antigen of BALB/c leukemia RL male 1. Systemic transfer of resistance with these clones was accomplished only when exogenous (rat or human) IL-2 was administered at the same time. Intraperitoneal injection of CTLL-A2 cells accelerated rejection of sarcoma Meth A (H-2Dd), but not ascites sarcoma BP8 (H-2k) or leukemia EL4 (H-2b) inoculated subcutaneously into C57BL/6 mice. CTLL-R5 cells were examined in local (Winn tests) as well as systemic transfer experiments. When mixed with leukemia cells before subcutaneous injection, they suppressed the growth of leukemia RL male 1 without exogenous IL-2. When injected intraperitoneally, CTLL-R5 cells inhibited the growth of subcutaneous grafts of leukemia RL male 1 only when exogenous IL-2 was administered at the same time. CTLL-R5 did not inhibit the growth of other radiation-induced BALB/c leukemias.

Characterization of interleukin 2-dependent cytotoxic T-cell clones: IV. Production of α, β and γ interferons and interleukin 2 by Lyt-2+ T cells

Abstract The production of α, β and γ interferons (IFN) and interleukin 2 (IL-2) by Lyt-2 + -dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2D d or the unique RL♂1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-α and -β. Induction of IFN-γ was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2D d+ ) and RL♂1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL♂1-specific cell line CTLL-RP, produced IFN-γ after induction with RL♂1. Production of IFN-α, β depended on IL-2, whereas production of IFN-γ did not, although addition of highly purified IL-2 increased IFN-γ production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-γ but not IFN-α, β. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2 + cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.

Characterization of IL-2-dependent cytotoxic T-cell clones: II. Cell-surface phenotypes, histochemical and ultrastructural properties☆

This report examines the histochemical staining patterns, ultrastructure, and cell-surface phenotypes of six antigen-specific T-cell clones. Histochemical analyses indicated that all cell lines expressed alpha-napthyl butyrate esterase characteristic of the monocytic isoenzyme, intense napthol AS-D chloroacetate reactivity characteristic of granulocytes, and were negative for leucoperoxidase, alkaline phosphatase, and Sudan black. Only one clone stained weakly for acid phosphatase. The esterase staining patterns became evident in newly established cell lines after growth for only 1 week in interleukin 2-conditioned medium. Ultrastructurally, the outstanding feature was numerous membrane-bound granules containing a complex-appearing globular material. The cell-surface phenotypes of the lines as determined by protein A-sheep red blood cell rosetting and indirect immunofluorescence was Ly-5+, T200+, Qa-5-, MAC-1-, and Lyt-1+,2,3- for the helper line and Lyt-1-2,3+ for the five cytotoxic lines. By quantitative absorption analyses, low levels of Lyt-1 antigens were detected on all examined cytotoxic lines. The results strengthen the view that long-term T-cell lines can retain normal T-cell characteristics while also expressing markers that are either absent or in undetectable levels on uncultured T lymphocytes. The presence of the esterases may be associated with an expanded functional role in the T-cell lines.

The same genetic locus directs differentiation-linked expression of endogenous retrovirus gp70 on thymocytes and spleen cells in the mouse

The major envelope protein of murine leukemia virus (MuLV), gp70, is known to be expressed on thymocytes of certain mouse strains which rarely, if ever, express complete virions (Tung et al. 1975a, b). The occurrence of this virally encoded protein was originally detected because in some strains it carries the antigen G~x (Tung et al. 1975a, Stockert et al. 1971). Classically G~x has been described as an antigen characteristic of the thymic stage of T-cell differentiation, analogous in this respect to TL antigens. In G~x + strains such as 129, G~x can be detected on the surface of thymocytes but not on spleen cells (Stockert et al. 1971). Some Glxmouse strains such as C57BL/6 (B6) express gp70 molecules lacking the G~x antigen on thymocytes (Tung et al. 1975b). A few strains exhibit no detectable gp70, G~x + or Glx -. An interesting example of this sort of mouse is the congenic 129-G~xstrain, in which the G~xphenotype of B6 has been crossed into the 129 strain (Stockert et al. 1975). This strain is also negative for the thymocyte-surface gp70 characteristic of B6 mice, thus demonstrating that the Gix + gp70 of 129 and the Gixgp70 of B6 are encoded at nonallelic genetic loci (Tung et al. 1975b). [In the 129 mouse the locus designated Gv-I probably contains the structural gene for Grx + gp70, since this locus is semidominant in genetic crosses (Stockert et al. 1971)]. As predicted by this model, the reciprocal congenic, B6-GIx ÷, expresses both species of gp70 on its thymocytes. By the use of broadly reactive hyperimmune anti-gp70 serum, we have found that spleen cells from 129, B6, and B6-GIx +, but not 129-G~x-, mice also express cell-surface gp70. The gp70 in spleen appears to be present on mature T cells and on at least one other cell type. The latter probably includes B cells, but conclusive evidence on this point is not yet available. In G~x + mice the occurrence of endogenously encoded gpT0 molecules on spleen cells seems to be controlled by the same genetic locus which determines the presence of Glx + gp70 on thymocytes. Our analysis of spleen-cell surfaces for expression of MuLV-gp70 molecules involved standard immunochemical methods. Spleen cells of 129, B6, and their congenic Gixand Glx + derivatives were surface-radioiodinated by the lactoperoxidase method and the cells lysed with detergents. A group-specific goat anti-

Characterization of IL-2-dependent cytotoxic T-cell clones. III. Inhibition of killing activity by monosaccharides.

The effects of monosaccharides on the cytotoxic activity of cytotoxic T lymphocytes (CTL) and three cloned long-term cytotoxic T-lymphocyte lines (CTLL) are compared. Uncultured CTL and clones CTLL-A2 and CTLL-A11 were derived from the peritoneal cavity of C57BL/6 mice immunized against the H-2Dd determinants on the BALB/c sarcoma Meth A. Clone CTLL-R5 was derived from spleen of (BALB/c X C57BL)F1 mice immunized against a unique determinant on the BALB/c radiation-induced leukemia RL male 1. The cell-surface phenotype of the clones is Lyt-1+,2+,3+. Cytotoxic activity of CTLL-A2 and CTLL-R5 as determined by a 4-hr 51Cr-release assay was inhibited over 50% by 1 mM 2-deoxy-D-glucose. CTLL-A11 and the uncultured cytotoxic T cells were more resistant to inhibition by 2DG (40% at 20 mM). Surprisingly, it was found that the addition of D-mannose, D-galactose, D-glucose, L-fucose, alpha-methyl-D-mannose, and N-acetyl-D-glucosamine also inhibited, in a dose-related manner, the cytotoxicity of CTLL-A2 and CTLL-A11. CTLL-R5 showed a more restricted inhibition pattern: only D-mannose and D-galactose were inhibitory. The mechanism of inhibition remains to be clarified.

Regulation of T-Cell Proliferation by Interleukin-2 in Male Homosexuals with Acquired Immune Deficiency Syndrome

In 1981, a sudden increase in the incidence of Kaposi’s sarcoma (KS), Pneumocystis carinii pneumonia, and other opportunistic infections was recognized among highly promiscuous homosexual men (Centers for Disease Control, 1981). More recently, these diseases are being observed among heterosexual men who are intravenous drug users, the sexual partners of these heterosexuals, and Haitian refugees (Centers for Disease Control, 1982). The disease has been named the acquired immune deficiency syndrome (AIDS).

INTERLEUKIN REGULATION OF THE IMMUNE SYSTEM (IRIS) IN MALE HOMOSEXUALS WITH ACQUIRED IMMUNE DEFICIENCY DISEASE SYNDROMEa

During the spring of 1981, hospitals in New York and California reported to the Centers for Disease Control in Atlanta a dramatic increase in the Occurrence of Kaposi’s sarcoma, Pneumocystis carinii pneumonia, and other opportunistic infections among a group of highly promiscuous homosexual men.’ More recently, these infections have been reported among heterosexual men who are intravenous drug users, their sexual partners, Haitian refugees, hemophiliacs, and possibly a few patients who have received blood transfusions.2 The syndrome has been named the acquired immune deficiency syndrome (AIDS). Several studies have described the T-cell dysfunction characteristic of patients with AIDS, as well as the groups at the greatest risk for developing AIDS, as an increase in the percentage of suppressor/cytotoxic T-cell subpopulations or as a lower than normal ratio of helper (OKT 4; Leu3) to suppressor (OKT 8; Leu2) cells? ’ Others reported absent mitogen responses and depressed natural killer cell activity from homosexuals with Kaposi’s sarcoma.b The etiological agent(s) responsible for the immunological defect@) are not known. Gallo et al.’ isolated, however, human T-cell leukemia virus (HTLV) from cultured lymphocytes of patients with AIDS, and Essex et al. showed that 19 of 75 patients with AIDS had antibodies reactive to surface antigen of HUT102, a HTLV infected human T-cell line.’ More recently, HTLV-111, a retrovirus, has been linked to the possible etiological agent of AIDS. We have studied a group of patients with AIDS and a group of patients with lymphoma. Both groups are deficient in their proliferative response to phytohemagglutinin (PHA) and in their production of both interleukin-2 (IL-2) and interferongamma (IFN-7). Addition of indomethacin restored the IL-2 and IFN-7 production to PHA in patients with lymphoma but not in patients with AIDS. Addition of IL2 partially corrected IFN-7 production and proliferative activity in the patients with AIDS.