Maurice K. Gately

Regulation of human cytolytic lymphocyte responses by interleukin-12

IL-12 is a heterodimeric cytokine which has been shown to cause the proliferation of activated T and NK cells, to enhance the lytic activity of NK cells, and to induce IFN-gamma production by resting and activated T and NK cells. We previously reported that IL-12 could synergize with IL-2 to activate human LAK cells in the presence of hydrocortisone but that IL-12 alone was inactive. We herein show that in the absence of hydrocortisone, IL-12 by itself can activate human LAK cells. IL-12-induced LAK cell activity was mediated predominantly by CD56+ lymphocytes. Activation of LAK cells by IL-12 appeared to be independent of IL-2 since it was not inhibited by neutralizing anti-human IL-2. However, IL-12- and IL-2-induced LAK cell activation could be partially inhibited by anti-human TNF-alpha. Moreover, IL-12 produced in situ appeared to play a role in IL-2-induced LAK cell activation since rat monoclonal antibodies to human IL-12 could partially inhibit the generation of LAK cells in response to IL-2. In addition to its effects on LAK cell responses, IL-12 could facilitate specific allogeneic human CTL responses. However, IL-12-facilitated CTL responses were blocked by neutralizing anti-human IL-2 indicating a requirement for IL-2 produced in situ. The ability of IL-12 to facilitate both nonspecific LAK and specific CTL responses suggests that it may be useful as a therapeutic agent against some tumors and infectious diseases.

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

Abstract A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro . The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro , one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.

The Role of Calcium in the Lethal Hit of T Lymphocyte-Mediated Cytolysis

The cytolytic T lymphocyte (CTL)1 inflicts lethal damage within minutes after contact with a specific antigen-bearing target cell. The mechanism of this damage remains a mystery. Elsewhere in this volume (1), we have reviewed progress during the past decade towards understanding the mechanism of CTL-mediated killing.

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. V. Evidence that manganese inhibits a calcium-dependent step in programming for lysis.

Abstract The mechanism of T-lymphocyte-mediated cytolysis consists of three successive steps: adhesion formation, programming for lysis, and killer-cell-independent lysis. Mg 2+ , but not Ca 2+ , is required for adhesion formation, whereas programming for lysis is strongly Ca 2+ dependent. We have previously reported that the transition metal manganese can substitute for Mg 2+ in supporting adhesion formation. In the present paper, we demonstrate that manganese inhibits programming for lysis. The inhibitory effect of Mn 2+ on cytolysis can be reduced by increasing the concentration of Ca 2+ . Furthermore, inhibitor sequencing experiments were unable to distinguish the step blocked by Mn 2+ from the Ca 2+ -dependent step. These results suggest that Mn 2+ blocks a Ca 2+ -dependent step(s) in programming for lysis. Present evidence does not distinguish whether the action of Ca 2+ in programming for lysis is via a Ca 2+ influx (as a “second messenger?”) or whether Ca 2+ simply serves as a cofactor at the cell exterior.

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.

p215 and p24: two membrane-associated proteins expressed on cloned cytolytic T-cells but not on cloned helper T-cells

Cloned lines of murine alloreactive cytolytic and helper T cells were derived from secondary mixed leukocyte cultures. Cells from three TC lines and four TH lines were internally labeled with 35S-methionine and then disrupted by hypotonic lysis. Low density (plasma membrane-enriched) and high density (endoplasmic reticulum-enriched) membrane fractions were isolated from each cloned cell line and analyzed by SDS-PAGE under reducing conditions. Two proteins were identified which were associated with membrane fractions from each of the TC lines but none of the TH lines. One of these, p215, migrated as a broad band with an apparent molecular weight of 200-220 kD. The other, p24, migrated as a sharp band or closely spaced doublet with an apparent molecular weight of 24 kD. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1, and Lyt 2 revealed that p215 was a variant of T200 found on TC lines but not on TH lines. Treatment of solubilized membrane proteins from TH lines with anti-T200 precipitated a 180-195 kD protein band seen on each of the TH lines but none of the TC. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It appears that p24 represents a previously unidentified protein which is unique to TC and thus deserving of further study as to its functional significance.

IL-2-Independent Activation of LAK Cells by a Heterodimeric Cytokine, Interleukin-12

Interleukin-12 (IL-12) is a heterodimeric cytokine which was originally isolated from cultures of activated human B lymphoblastoid cells and called natural killer cell stimulatory factor (Kobayashi et al., 1989) or cytotoxic lymphocyte maturation factor (Stern et al., 1990). IL-12 has been shown to stimulate the proliferation of activated T cells and natural killer (NK) cells (Gately et al., 1991) and to cause interferon-γ (IFN-γ) production (Kobayashi et al., 1989; Chan et al., 1991) and enhanced NK lytic activity (Kobayashi et al., 1989; Wolf et al., 1991) by resting peripheral blood mononuclear cells (PBMC). IL-12, unlike IL-2, does not cause resting PBMC to proliferate, but it can stimulate enhanced proliferation of PBMC cultured in suboptimal concentrations of IL-2 (Gately et al., 1991). IL-12 is composed of two disulfide-bonded subunits with molecular masses of 35 and 40 kDa (Kobayashi et al., 1989; Stern et al., 1990). The cDNA encoding each of these two subunits has recently been cloned and bioactive recombinant IL-12 (rIL-12) expressed (Gubler et al., 1991; Wolf et al., 1991). Coexpression of the two subunits is required for biologically active IL-12 to be produced (Gubler et al., 1991: Wolf et al., 1991).

Comparison of membrane-associated proteins of murine cytolytic and helper cloned T-cell lines: identification of a protein, p24, prominent in membrane fractions from cytolytic but not helper T-cells.

It has recently been reported that liposomes containing membrane components from cytolytic T-cell (TC) clones could transfer lytic activity to noncytolytic T- and B-cell lines, strongly suggesting that TC possess membrane-associated molecules which noncytolytic lymphocytes lack and which play a critical role in the lytic mechanism. It was thus of interest to compare the membrane-associated proteins from TC-lines to those of noncytolytic helper T-cell (TH) lines to determine whether any membrane-associated proteins unique to TC could be identified. Cells from three TC-lines and four TH-lines were internally labelled with [35S]methionine and then disrupted by hypotonic lysis. Low-density (plasma membrane enriched) and high-density (endoplasmic reticulum enriched) membrane fractions were isolated from each cloned cell line and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two proteins were identified which were prominent in the membrane fractions from each of the three TC-lines but not in the membrane fractions from any of the four TH-lines. One of these, p215, migrated as a broad band with an apparent mol. wt of 215,000. The other, p24, migrated as a sharp band, or tightly spaced doublet, with an apparent mol. wt of 24,000. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1 and Lyt 2 suggested that p215 was a variant of T200 found on TC-lines but not on TH-lines. Treatment of solubilized membrane proteins from TH-lines with anti-T200 precipitated a 185-kD protein seen on each of the TH-lines but on none of the TC-lines. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It therefore appears that p24 represents a previously unidentified protein which is strongly expressed by TC but not by TH and is thus deserving of further study as to its functional significance.