Guy D. Bonnard

Natural killer cells. Characteristics and regulation of activity.

Recently there has been increasing recognition of natural cell-mediated cytotoxicity as a potentially important antitumor effector mechanism in addition to that of specifically immune T cells and of activated macrophages. Although natural cellular cytotoxicity was first recognized only a few years ago (Herberman et al. 1973,1974, McCoy et al. 1973b, Oldham et al. 1973, Rosenberg et al. 1972,1974), there has already been extensive research in many laboratories on the nature of the effector cells, the possible mechanisms of cytotoxicity, the factors regulating the levels of reactivity, and the relevance of natural immunity to in vivo resistance against tumor growth and immune surveillance. A principal component of natural cell-mediated cytotoxicity in rodents and man has been found to be a particular subpopulation of lymphocytes which have been termed natural killer (NK) cells. We have recently reviewed in detail much of the available information on NK cells (Herberman & Holden 1978). In this paper we will only summarize our views on the characteristics of NK cells and focus on a few issues of current interest in our laboratory.

Continued growth of functional human T lymphocytes: production of human T-cell growth factor.

Abstract Interest in the possibility of growing large numbers of antigen-reactive human T lymphocytes led to a thorough study of the production of T-cell growth factor(s) that allow the continued proliferation of human T lymphocytes in culture. Such factors are found in the supernates of human peripheral blood lymphocytes stimulated with phytohemagglutinin. Whereas the growth factor from a commercial laboratory was produced with pooled lymphocytes from many donors, we developed supernates from mononuclear peripheral blood leukocytes of single donors. The present study has revealed several important points for production of T-cell growth factor: (i) the commercial production was unnecessarily complex, and could be simplified; (ii) stimulation of leukocytes with phytohemagglutinin was needed, and could not readily be replaced by Con A or alloantigens; (iii) some normal donors were high producers of T-cell growth factor, whereas others were consistently low producers; (iv) adherent cells, probably monocytes, could be suppressive in this system. A synergistic effect between the stimuli of phytohemagglutinin and of cells from certain B lymphoblastoid cell lines was also found.

Cultures of purified human natural killer cells: Growth in the presence of interleukin 2

Abstract We have isolated highly enriched populations of LGL, which are virtually devoid of mature typical lymphocytes (as enumerated by morphological and surface antigen analysis using monoclonal antibodies e.g., OKT3) in comparison to T cells which contain greater than 95% sheep erythrocyte-forming cells and are devoid of LGL and NK K activities. Both types of cells grew in the presence of crude or partially purified IL-2. Cultures of LGL could be initiated consistently even in the absence of lectins and the cultured LGL retained their characteristic morphology and cytotoxic activity. However, within 7–10 days after initiation, the cultured LGL changed in surface phenotype to become antigenically indistinguishable from cultured T cells.

A rapid technique for isolation of viable tumor cells from solid tumors: Use of the tumor cells for induction and measurement of cell-mediated cytotoxic responses

A rapid and simple technique for the isolation of viable tumor cells from human and mouse solid neoplasms is described. It consists of a 5 to 10-min treatment with trypsin-collagenase-DNase mixture, followed by mechanical disaggregation of the tumor tissue and subsequently by a brief centrifugation on a discontinuous Percoll gradient. With the tumors employed, this procedure usually requires less than 1 hr and results in preparations comprising greater than 80% tumor cells with viability of 80-90%. Cell-mediated cytotoxic response was measured with: (a) unsensitized lymphocytes freshly obtained from tumor-bearing hosts; (b) lymphocytes propagated in culture with T cell growth factor; and (c) lymphocytes stimulated in cocultures with autologous or syngeneic tumor cells. The cytotoxic activity was assessed in a modified [51Cr]-release assay adapted for solid tumor cells, allowing a long incubation period (24 hr) and the use of a low number (200-1000) of highly labeled target cells (2-10 counts/min/cell).

Human tumour antigens defined by cytotoxicity and proliferative responses of cultured lymphoid cells

The long-term goal of many laboratories has been to develop cellular reagents having specific reactivity against human tumour cells. Such immune cells should prove useful for defining the antigenicity of human malignancies and may have important therapeutic potential, as has been clearly shown in some animal models1. Here we describe methods of initiating continued lymphocyte cultures (CLC) having specific anti-tumour reactivity using conditioned media containing interleukin-2 (IL-2).

Modulation of glucocorticoid receptors by mitogenic stimuli, glucocorticoids and retinoids in normal human cultured T cells☆

Human T lymphocytes can be maintained in cell culture by the addition of conditioned medium (CM) containing a T cell growth factor (TCGF). This system provides an opportunity to study the presence and modulation of glucocorticoid receptors (GR) by various factors in these cells. GR were present in T cells grown from each of 22 normal individuals; the binding capacity (mean = 3851 +/- 2880 sites/cell) and affinity (Kd = 7.4 X 10(-9)) were similar in rested cultured T cells (CTC) to those reported in peripheral blood T lymphocytes. Treatment of rested CTC with stimuli such as phytohemagglutinin (PHA), CM, or 12-O-tetradecanoylphorbol-13-acetate (TPA) results in a mean increase in GR binding capacity of 3.1-, 3.2- or 2.4-fold respectively without modification of binding affinity. Using an exchange assay to measure occupied and unoccupied GR, we examined the effects of cortisol on its own receptor. Treatment of rested CTC with 10(-7) M cortisol for 24 h decreased GR by more than 50%. Cortisol treatment also blocked the induction of GR by PHA and CM. Since retinoids have been shown to modulate the immune response and to alter the effects of PHA and phorbol esters on lymphocytes, we examined their effects on GR. Retinol decreased GR activity in CTC but only at concentrations which inhibit cell growth. It is concluded that GR activity in human T cells can be modulated by several important factors involved in lymphocyte function.

Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells: II. Evidence that Kilham rat virus is responsible for the inhibitory effect

Abstract Kilham rat virus (KRV) was isolated from the lymphocyte cytopathic 13762 summary adenocarcinoma tumor line described in part I of this report, as well as three other in vivo passaged rat tumors maintained in the same animal room. It could not, however, be isolated from the noncytopathic (CS8NT)D tissue culture line. Tests done with CsCl 2 -purified KRV preparations showed that the virus could replicate in rat lymphocytes and could profoundly depress lymphocyte viability and lymphoproliferative responses. Heterologous anti-KRV antiserum could reverse the inhibitory effects of the purified virus preparation and the inhibitory effects of ultrasonically disrupted KRV-infected tumor cells, but could only partially reverse the inhibitory properties of X-irradiated whole 13762 tumor cells. The results suggest that KRV could account for some, if not all, of the inhibitory properties of the 13762 tumor line.

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation.

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells. As expected, elimination of class II HLA-positive AC with anti-class II HLA MoAb plus complement caused a decrease in proliferation of CTC in response to all the mitogens tested. In contrast, elimination of class II HLA-positive CTC was shown to clearly increase proliferation of CTC, perhaps because this may deplete class II HLA-positive suppressor cells.

Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infectious agent

Abstract Small numbers of X-irradiated 13762 cells added as third-party cells to mitogen response assays or mixed lymphocyte cultures caused a significant reduction in viability of the cocultivated lymphocytes, and completely inhibited the expected lymphoproliferative responses. Results showed that the factor(s) responsible for the inhibitory effect was preserved after ultrasonic disruption of the tumor cells, could be sedimented by ultracentrifugation, and was sensitive to treatment with ultraviolet light. Further, cytopathic effects could be serially propagated using cell-free supernatants obtained from sonicated 13762 tumor cells. The results suggest that the 13762 adenocarcinoma line, as carried in vivo in this laboratory, harbors an infectious particle which can affect the proliferative responses of lymphocytes in vitro .

Antitumor reactivity in vitro and in vivo of lymphocytes from normal donors and cancer patients propagated in culture with T cell growth factor (TCGF)

Abstract Peripheral blood lymphocytes (PBL) from 38 normal donors and from 27 cancer patients were propagated in bulk cultures for 3–6 weeks using T cell growth factor (TCGF). In addition, cultures derived from lymphocyte preparations enriched for or depleted of natural killer (NK) cells and several clones of cultured cells were studied. The following main observations were made: (a) PBL of both patients and healthy donors could be expanded to large numbers (up to 2500-fold); (b) CLC derived from unfractionated PBL exhibited intermediate levels of cytotoxic activity against autologous and allogeneic fresh lung tumor cells and strong cytotoxicity toward several cultured adherent tumor cells; (c) whereas cultures originated from populations enriched for NK cells were highly cytotoxic against both adherent tumor target cells and against an NK-sensitive leukemic cell line (K562), cultures derived from populations depleted of NK cells were preferentially cytotoxic to adherent target cells; (d) clones of CLC were also strongly cytotoxic, but 2 out of 3 clones tested showed a narrower spectrum of target cytotoxicity than that of uncloned CLC; (e) CLC, when mixed with two carcinoma cell lines, were able to inhibit tumor growth in nude mice.