Kurt Berg

The effect of interferon on lymphocyte-mediated effector cell functions: Selective enhancement of natural killer cells

Abstract The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target.

Measurements of changes in histocompatibility antigens induced by interferons.

Publisher Summary This chapter discusses the measurements of changes in histocompatibility antigens induced by interferons. A major nonantiviral effect, among others, of interferon is the modulation of histocompatibility antigens on the surface of a variety of cells. Concerning major histocompatibility complex (MHC) class 1 antigens (HLA-A, -B, and -C), all three types of human interferons (Hu-IFNs) (alpha, beta, and gamma) induce an increase in expression and synthesis of these antigens. When looking at MHC class II antigens, Hu-IFN-γ is the only type of interferon able to induce a substantial increase in the expression of these antigens, and cells from the monocyte/macrophage lineage are, especially sensitive to this effect of immune IFN. Several methods have been reported for the demonstration of MHC enhancement, but an indirect immunofluorescence method with subsequent measurement in a fluorescence-activated cell sorter (FACS-II) is found to be easy, convenient, and sensitive to very small changes. By storing the data in the FACS, it is possible to recall them in a computer system for further calculations.

Natural killer cell activity correlates with the amount of β2-microglobulin on human peripheral blood lymphocytes

Abstract Fractionation of human peripheral blood lymphocytes according to either their size or their amount of β 2 -microglobulin ( β 2 -M) or HLA molecules in the fluorescence-activated cell sorter has enabled us to test the possible relationship between the NK activity, on the one hand, and the amount or the density of surface antigens on the other. Generally, large lymphocytes exhibited higher NK activity than the small ones, and likewise, lymphocytes with high content of β 2 -M were better NK effectors than the low β 2 -M containing cells. However, since small lymphocytes with high β 2 -M content were better NK effectors than large lymphocytes with low β 2 -M content, the above-mentioned findings were relative rather than absolute. In contrast to the close relationship between β 2 -M and NK activity, the amount of HLA antigens (measured with a monoclonal anti-HLA framework antibody) was not found to be correlated to NK activity. The notion that β 2 -M was important in the NK reaction was supported by experiments using papain-treated effector cells which showed that a selective removal of β 2 -M from the cell surface was paralleled by a decrease in the NK reactivity. However, since F(ab′) 2 fragments of the anti- β 2 -M antibody did not inhibit NK activity, the β 2 -M molecules itself cannot be a part of the NK receptor. Finally, regarding the enhancement of NK by interferon, it was shown that lymphocytes with high amounts of β 2 -M were more prone to this effect than lymphocytes with low amounts of β 2 -M.

EFFECTS OF INTERFERON ON HUMAN LYMPHOID CELLS AND THEIR FUNCTION

It was already shown some years ago in mice that interferon exerts nonantiviral effects on lymphoid cells in vitro as well as in vivo. In parallel with progress in procedures to purify human interferon (IF), and with the acceleration of clinical use of human interferon, growing interest in the various biological effects exerted by the compound is becoming apparent. The influence on cells of the lymphoid apparatus in man will in this context stand as a subject of major importance and the present paper will deal with this. We have made observations in three different experimental systems. One includes methods to quantitate the amounts of major histocompatibility antigen (MHC) expressed on the membrane of lymphocytes and monocytes, another the assessment of natural killer cell (NK) function, and the third is a system where human lymphocytes become stimulated during mixed lymphocyte reactions (MLR) to develop alloaggressive cytolytic effector T cells that are assessed in a cell-mediated lympholysis assay (CML). In the two former systems different species of completely pure human leukocyte interferons have been assessed. Complete purification of leukocyte interferon was accomplished as described in previous papers., Partially purified leukocyte interferon (PIF) with a specific activity of 1 x 10 IFU/mg was a generous gift from Dr. K. Osther, Copenhagen. This material was used for further purification. FIGURE 1 shows the stained protein bands with an input of 1 x 10 international interferon units (IFU) on the SDS gels. The upper curve of the FIGURE gives the antiviral (a-v) activity recovered from gel slices; it is apparent that the a-v activity appears in two major molecular species, and three additional minor species are disclosed by the analysis.

SIZE DISTRIBUTION AND β2-MICROGLOBULIN CONTENT OF HUMAN NATURAL KILLER CELLS AS ANALYZED BY FLUORESCENCE ACTIVATED CELL SORTING (FACS)1

Publisher Summary This chapter focuses on size distribution and β2-microglobulin content of human natural killer (NK) cells analyzed by fluorescence-activated cell sorting (FACS). In a study described in the chapter, normal peripheral blood mononuclear cells were isolated by Isopaque–Ficoll method and stained with a FITC-conjugated rabbit anti-β2-M antiserum. The cells were subsequently processed on a FACS II machine. In the chapter, the dot plot screen of the FACS II from a typical experiment is shown, each cell being represented by a dot giving its size (or forward light scatter, x-axis) and the fluorescence intensity (y-axis). Both lymphocytes and monocytes—as verified by Giemsa-stained smears—can be delineated and lymphocytes can be further subdivided into four lymphocyte subsets according to their β2-M content and size. The FACS was set up to sort the 20% least and the 20% most fluorescing cells, and it was found that cells with low β2-M content, irrespective of size, always exhibited lower NK than the high β2-M containing cells and that IFN-inducible NK cells moreover were found in the β2-M rich cell fractions, indicating that the association between β2-M and NK might be stronger than that between NK and cell size.