Synergistic cytotoxicity. II. In vitro arming of monocytes and T cells by a heat labile fraction of human plasma.

In a previous paper we demonstrated that freshly obtained human plasma contain a heat labile nonantibody factor that induced human mononuclear cells to become nonspecifically cytotoxic toward xenogeneic but not allogeneic RBC targets. We now present evidence that this factor has a loose affinity for human monocytes and human T cells and can arm then to kill xenogeneic RBC targets. Furthermore, proteolytic enzymes markedly enhance this arming effect. This ability to be armed by a heat labile component found in fresh human plasma and the fact that proteolytic enzymes markedly enhance cytotoxicity clearly dissociate this model of nonspecific cytotoxicity for previously reported NK models.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. I. Activation by tuberculin and by Staphylococcus filtrate

Normal lymphocytes activated by mitogens such as phytohemagglutinin (PHA), by Staphylococcus filtrate (SF), or lymphocytes from sensitized individuals stimulated by antigen (PPD, etc.) are cytotoxic to tissue culture cells of different origins. In this and the following paper, the results of a detailed quantitative analysis of the specificity of this cytotoxic reaction are presented. Effector cells were human or mouse lymphocytes, activated by PHA, SF, PPD, or serum factors in the culture medium. Cells from established cell lines of human, mouse, hamster, or rabbit origin, or primary human or rat embryonic fibroblasts were used as target cells. Lysis was quantitated by release of ⁵¹Cr from labeled target cells.Purified human blood lymphocytes, activated by PPD, SF, or otherwise, preferentially damaged allogeneic target cells. Lysis of xenogeneic target cells was weak or did not occur. A close correlation was noted between target cell destruction and blastoid transformation of the lymphocytes, but the slope of the regression lines of xenogeneic cytotoxicity was much smaller than that of allogeneic cytotoxicity when plotted as a function of blastoid transformation.Lymph node or spleen cells from CBA mice were stimulated by PPD to transformation and DNA synthesis. CBA lymphocytes also showed an increased degree of blast transformation in medium containing fetal calf serum or certain batches of fresh human serum. Mouse lymphocytes activated in these ways damaged allogeneic L cells but had no effects on xenogeneic Chang cells.These results indicate that lymphocytes activated by various means preferentially damage target cells from their own species. The recognition mechanisms which determine the specificity of the reactions are not known.     

Nonspecific cytotoxic effects of antigen-transformed lymphocytes. III. Relationship to specific cytotoxicity.

Human peripheral blood lymphocytes, cultured either with PPD or in one-way mixed leucocyte reactions, develop a nonspecific cytotoxic potential which can be expressed on autologous as well as on unrelated xenogeneic target cells. This nonspecific cytotoxicity develops at an early stage in the mixed leucocyte reaction, before specific cytotoxic effects are demonstrable, and decreases as specific cytotoxicity increases.     

Effect of serum on autologous cell recognition by macrophages

The non-immune mechanisms of recognition of self and non-self substances by macrophages has not yet been clarified. In this work, we report the ability of mouse peritoneal macrophages to attach to and phagocytize in vitro autologous and homologous erythrocytes in proportions as high as those for certain heterologous red blood cells. This ability was abrogated by autologous or homologous serum but not by heterologous serum or a serum-free supplement. This effect of serum was dose dependent and did not affect the phagocytosis of homologous "old" red cells. Procedures for the identification of this serum factor indicated that it was dialyzable (10 kD cut off) and was excluded by filtration in Sephadex G-25. We conclude that this finding supports the possibility that macrophages do not selectively phagocytize foreign particles or senescent cells but, rather, that they do phagocytize all particles or cells indiscriminately and this serum factor may prevent phagocytosis of normal self cells.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Intracellular localization of anti-tumor immune RNA.

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.     

In vitro allogeneic cytotoxicity in the solitary urochordate Styela clava.

Hemocytes from the solitary urochordate Styela clava can effect allogeneic cytotoxicity in vitro. Spectrophotometric and microscopic quantification of eosin-y dye exclusion revealed significantly greater frequencies of cell death in allogeneic hemocyte cultures when compared to autogeneic controls. This cytotoxic response was characterized by 1) transient activity such that specific cytotoxicity could be detected for only 4 hours of culture though continued specific killing may have been obscured by spontaneous cell death; 2) a necessity for cellular interaction demonstrated by the elimination of allogeneic cytotoxicity in the absence of cell contact; 3) killing of multiple targets by effector cells due to high levels of response at low allogeneic ratios; 4) insensitivity to altered temperature; 5) increased cytotoxicity in the absence of autologous plasma; 6) an absence of xenogeneic reactivity; 7) the presence of three hierarchical levels (low, intermediate, and high) of response. These data reflect events involved in the recognition of allogeneic cellular determinants resulting in specific cytotoxicity effected by immunocompetent cells. Such in vitro recognition and cytotoxic recognition and cytotoxic reactivity may be responsible for adaptive reactions caused by histoincompatibility in solitary tunicates.     

Lymphocyte cytotoxicity to influenza virus-infected cells. II. Requirement for antibody and non-T lymphocytes.

Peripheral blood lymphocytes (PBL) obtained from humans were cytotoxic for influenza virus-infected target cells. The PBL were shown to have associated influenza virus anti-hemagglutinin antibody (AHAb) detectable only by radioimmunoassay. This antibody could be removed by incubating PBL at 37 degrees C for 30 min. The lymphocyte population that was effective in this system was nonadherent and nonphagocytic cells. PBL gave comparable levels of cytotoxicity when tested by using either a xenogeneic or allogeneic virus-infected target cell. These results indicate that lymphocyte cytotoxicity to influenza virus infected cells may be mediated by small quantities of antibody and by lymphocytes that possess characteristics of K cells. No evidence for T cell-mediated cytolysis was found with this xenogeneic system.     

Role of soluble cytotoxic factor in concanavalin A-induced cellular cytotoxicity to rabbit erythrocytes.

A new sensitive and simplified method for detecting concanavalin A-induced cellular cytotoxicity was developed by using 51Cr-labeled rabbit erythrocytes as target cells, and a soluble cytotoxic factor (SCF) to rabbit erythrocytes was demonstrated with this system. From morphological studies, this factor appeared to play a major role in cellular cytotoxicity. The cytotoxic activity of SCF was removed by absorption with rabbit erythrocytes. Furthermore, the target cells pretreated with SCF and washed were subsequently destroyed. These results may indicate that SCF exerts its effect through binding to cellular receptors. Some monosaccharides showed inhibitory effects on the cytolysis of rabbit erythrocytes by SCF and on the adsorption of SCF activity to them. The significance of these findings in elucidation of the action of SCF was discussed.     

Natural killer cells do not lyse resting or mitogen-stimulated B cells

It has recently been reported that isolated resting natural killer cells lyse autologous resting and mitogen-stimulated B cells. In this report, we have been unable to corroborate these observations and provide indirect evidence that lytic susceptibility is attributable to exposure of the target cells to xenogeneic antigens present in fetal calf serum (FCS). Moreover, we show that interleukin-2-activated killer cells potently lyse normal peripheral blood mononuclear cells which are exposed to FCS.     

Nonspecific human lymphocyte-mediated cytotoxicity induced by immobilized IgG aggregate

Normal human lymphocytes were induced to lyse nonsensitized erythrocytes when concomitantly incubated on immobilized IgG aggregate with various erythrocyte target cells. These included ox, sheep, chicken, and human red blood cells. Only immobilized aggregate would initiate cytolysis. The IgG aggregate was prepared from normal, healthy adult donors and did not possess target cell specificity (e.g., human erythrocyte lysis was initiated by autologous IgG). Normal human lymphocytes could be induced to lyse the red blood cell targets only after a preincubation with adherent mononuclear cells; however, freshly prepared lymphocytes depleted of IgG-FcR^? cells were cytolytic. Cytolysis induced by immobilized IgG-aggregate can be distinguished from NCMC and ADCC by its requirement for immobilized IgG aggregate and the absence of target cell specificity in the IgG-aggregate preparation.     

Generation of cytotoxic lymphocytes in the autologous mixed lymphocyte culture.

We have studied the ability of purified B lymphocytes to generate cytotoxic T lymphocytes in autologous mixed leukocyte cultures (MLC). Cytotoxic lymphocytes were produced but only autologous mononuclear cells stimulated by lipopolysaccharide (LPS) were susceptible target cells. Unstimulated mononuclear cells and purified B cells were not susceptible to killing by cytotoxic cells generated in the autologous MLC. This suggests that the target antigen may be expressed on stimulated or dividing B lymphocytes in a way that renders the cells more susceptible to cytolysis. Autologously stimulated cytotoxic effector cells were found to exhibit specificity. Cy totoxicity for autologous LPS-stimulated target cells occurred but not for an allogeneic, B cell, histiocytic lymphoma cell line. It is postulated that cytotoxic T cells generated in the autologous MLC may play a role in immune surveillance or in regulation of the immune system.     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

Sera from lipopolysaccharide (LPS)-injected mice exhibit complement-dependent cytotoxicity against syngeneic and autologous spleen cells.

To determine if lymphocytes are able to discriminate between self and nonself, the polyclonal B-cell activator lipopolysaccharide (LPS) was injected into mice, and sera from those mice were tested at different times for their cytotoxic effect against autologous and syngeneic isotope-labeled spleen cells in the presence of complement. It was regularly found that LPS caused the appearance of cytotoxic activity in sera detectable against autologous and syngeneic spleen cells. This cytotoxicity was found to be complement dependent, and it was abolished by absorbing the sera with the target cells. LPS did not induce cytotoxic serum activity in the LPS nonresponder strain C3H/HeJ. When the serum was passed through an anti-mouse Ig column, the eluted sample completely lost its cytotoxicity. It is likely, therefore, that these cytotoxic factors are immunoglobulins with specificity for self, suggesting that tolerance to thymus-dependent autoantigens does not exist at the B-cell level. The implications of this possibility for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.     

Antibody in the sera of tumor-bearing mice that mediates spleen cell cytotoxicity toward the autologous tumor.

Pretreatment of MTV-induced BALB/cfC3H mammary tumor cells with autologous serum results in increased spleen cell cytotoxic activity and the recruitment of previously inactive spleen cells to cytotoxic activity against the target cells. These recruiting antibodies are tumor-specific for individual tumors; pretreatment with such serum of target cells of an MTV-induced mammary tumor obtained from a different BALB/cfC3H female results in blocking of spleen cell activity. The autologous recruiting factors are active at dilutions of 1000 or more of whole serum are found in the 19S fraction after gel filtration.     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.     

Induction of autoreactive cells by the preculture of human peripheral blood mononuclear cells with the autologous fresh plasma.

An AMLR in which precultured cells proliferated in response to fresh non-T cells is described. In our system, the responder is human peripheral blood mononuclear cells precultured in the autologous fresh plasma for up to 16 days, and the stimulator is fresh autologous non-T cells. Results suggested that there were two subpopulations of autoreactive cells obtained from the preculture; the high and low density small lymphocytes, both having ERF activity. The autoreactivity of low density cells was augmented when either macrophages or N-ERF-cells were depleted from PBM and thereafter precultures wre performed. A survey of the functional characteristics of the responding cells showed that the responding cells had NK activity against Molt-4 cells but had no significant ADCC activity against target CRBC. Mechanisms for the induction of autoreactive cells by the preculture in the presence of autologous fresh plasma are discussed.     

Alloantibody-induced cytotoxicity of macrophages.

Alloantibodies substantially alter the in vitro nonspecific cytotoxic effect of macrophages on bystanding allogeneic and xenogeneic target cells. Although the specific IgM alloantibody increased significantly the ability to parental macrophages to damage target cells, IgG alloantibody had an opposite effect and suppressed the macrophage cytotoxicity. Macrophages of F1 hybrid mice were less affected by this treatment unless alloantibodies against both parental strains were added together. Parental macrophages exposed in vitro to the concomitant action of both IgM and IgG alloantibodies exhibited a diminished cytotoxicity toward target cells. It is proposed that IgM and IgG alloantibodies induce different conformational changes of the macrophage surface.     

An assay for serum cytotoxicity against erythroid precursor cells in pure red cell aplasia

Several reports have indicated that a circulating serum inhibitor (antibody) is involved in the pathogenesis of acquired pure red cell aplasia (PRCA). In the present study, the pathophysiologic significance of this inhibitor was assessed according to the status of erythroid progenitor cells in the bone marrow. So far, direct proof for the antibody acting against erythroid stemcells was lacking. Employing an "in vitro" assay, erythroid colony forming cell (CFU-e) numbers in PRCA marrow were quantified and the cytotoxic effect of PRCA serum on CFU-e was investigated. It was revealed that the CFU-e population size in the marrow of PRCA patients was severely reduced; at the same time the relative number of myeloid colony forming cells was normal. The serum was demonstrated to contain a factor cell which was cytotoxic to CFU-e, in the presence of complement. The results indicate that inhibition of erythropoiesis in PRCA is achieved by a complement dependent plasma factor which eliminates or inactivates CFU-e and which constitutes an effective block at the precursor cell level in the differentiation pathway of the erythroid line. The data present a practical assay for measuring cytotoxic factors affecting erythroid stem cells.