Prekiller Cells Research Articles

Autologous and allogenic natural cell mediated cytotoxicity at the single cell level: divergent effects of interferons and interleukin 2 on binding and lysis.

In order to evaluate their divergent effects on binding and lysis of the NCMC, rH IFN-alpha and rH IL-2 were used for the in vitro-preincubation of normal donors' PBL, which were then tested as effector cells against K562 and the long-term cultured melanoma cell line RIMA in the SCCA. Both lymphokines significantly augmented the cytolysis of K562 without relevant influence on the conjugate formation. However, against RIMA IFN-alpha additionally amplified the binding affinity. This in keeping with the other authors' opinion that IFNs have target-specific effects at the single cell level, with the principal activation of already conjugated pre-killer cells against all the target cell lines tested. We performed a therapy-follow-up of the i.p. administration of rH IFN-gamma to patients with ovarian carcinomas in vivo. With the SCCA we detected a significant correlation of the duration of therapy with the autologous cytotoxicity in the ascitic compartment. In one case the increase of this parameter was even exponential. However, the countercurrent trend of the conjugate formation delayed and reduced the activation of the autologous total killer activity. This relative stagnation resulted in the inadequate clinical response of two patients. Moreover, we observed a discrepancy in the development of the autologous and allogeneic SCCA-parameters suggesting strong effects of the peritumorally administered IFN directly on the effusion tumor cells.

Isolation and characterization of protective T cells induced by Listeria monocytogenes.

Rats convalescing from a recent infection with Listeria monocytogenes generate T cells which can protect recipient rats against a challenge infection with that organism. Using monoclonal antibodies that react with some but not all rat peripheral T cells, the T-cell mediators of acquired resistance to infection (TCRI) were isolated by panning and characterized by using a fluorescence-activated cell sorter. Many L. monocytogenes-immune TCRI were relatively large cells as judged by their light-scattering properties. That finding accords with previously reported cytokinetic data and velocity sedimentation analyses which revealed that the majority of L. monocytogenes-immune TCRI are lymphoblasts. In the current investigation, the surface antigenic profile of these mediator T cells was revealed as W3/25+ OX8+ OX4+ RT6.1-. That phenotype is identical to that of L. monocytogenes-induced prekiller cells which are formed as part of the animal's cell-mediated response to infection. Like prekiller cells and their differentiated counterparts, L. monocytogenes-immune TCRI adhere preferentially to monolayers of syngeneic fibroblasts. The results indicate that L. monocytogenes-immune TCRI belong to a minor subset of peripheral T cells which also contains T cells that have the cytotoxic potential by which L. monocytogenes-induced prekiller cells have been defined.

Antiautologous lectin-dependent cell-mediated cytotoxicity: Evidence for prekiller cells as effector cells

Spleen cell killing of target cells can manifest through spleen cell-target cell interaction in the presence of mitogenic lectin, lectin-dependent cell-mediated cytotoxicity (LDCC). Spleen cells from C57B1/6 mice immunized with C3H mouse cells were found to be capable of cytotoxicity against autologous and other C57B1/6 spleen cells in the presence of Con A. Thus, alloimmune spleen cells are capable of an anti-self cytotoxic response in the presence of mitogenic lectin, antiautologous LDCC. Antiautologous LDCC is blocked by preincubation of cytotoxic cells with colchicine, an inhibitor of the cytotoxic effector mechanism. Analysis of alloimmune spleen cell subpopulations suggests that the antiautologous LDCC cell is an immature alloimmune cytotoxic cell (prekiller cell). Potent LDCC was found in alloimmune spleen cell preparations depleted of alloimmune cytotoxic T cells (killer-depleted) by three passes on allogeneic cell monolayers genetically identical with the immunizing cell. However, some LDCC effectors were also found to adhere to the adsorbing target, suggesting that there is some maturational diversity among LDCC effectors.

Decline, in aging mice, of the anti-2,4,6-trinitrophenyl (TNP) cytotoxic T cell response attributable to loss of Lyt-2-, interleukin 2-producing helper cell function.

The in vitro generation of cytotoxic T lymphocytes (CTL) specific for 2,4,6-trinitrophenyl (TNP)-modified syngeneic spleen cells is found to be almost invariably depressed in apparently healthy 18-month-old mice of the long-lived (BALB/c x C57BL/6)F1 hybrid strain. Studies of CTL production from Lyt-2+ thymus cells have suggested that pre-killer cells may require, for maturation into effectors, the presence of a soluble helper factor, interleukin 2 (IL2), produced by Lyt-2- cells which are themselves devoid of pre-CTL activity. We have therefore developed a petri-dish adherence technique for separating spleen cells into Lyt-2+ and Lyt-2- populations in order to test for helper and pre-killer activity independently. Pre-CTL function is measured by stimulating Lyt-2+ cells in the presence of exogenous IL2. Helper cell activity is tested by adding Lyt-2- cells to "indicator" populations of Lyt-2+ pre-CTL. Estimation of IL2 levels in medium conditioned by unfractionated, TNP-self-stimulated splenocytes provides a second measurement of helper cell function. Mice 18 months of age, when compared to 4 month-old controls, are found to retain nearly all of their pre-CTL activity, but to have lost sufficient helper cell activity to account for the decline in unseparated spleen cell cultures. Older mice also produce lower IL2 levels.

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes.

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.

The origins of alloreactivity: differentiation of prekiller cells to viral infection results in alloreactive cytolytic T lymphocytes.

Mice maintained in our animal colony become primed to Sendai virus. This "environmental" priming is reflected in a shift in prekiller activity from the Ly 123 to Ly 23 T cell set and in increased virus-specific cytolytic activity. This transition is accompanied by the development of cytolytic activity against allogeneic targets (not expressing Sendai antigens). These findings are consistent with the view that continued stimulation of Ly 123 cells by autologous MHC antigens, associated with foreign antigens such as a virus, generate Ly 23 prekiller cells that respond to alloantigens as well as autologous cells infected with the relevant virus.

IN VIVO GENERATION AND REGULATION OF KILLER T‐CELLS DIRECTED TOWARDS HAPTEN‐COUPLED SYNGENEIC CELLS*

We have extended our studies which show that hapten-specific killer T-cells can be generated in vivo towards hapten-conjugated, syngeneic spleen cells provided an auxiliary antigenic stimulus is incorporated. M1s antigen served in this auxiliary capacity by stimulating helper T-cells. Natural or experimental tolerance to these antigens abrogated cytotoxicity. Effecting tolerance in host animals towards target hapten also prevented development of the genetically restricted killer T-cells. Thus, cytolytic T-cells can be controlled equally at the levels of helper and pre-killer cells. While adoptive transfer of hapten-specific tolerance by means of spleen cells did not abrogate appearance of hapten-specific killer T-cells, it did prevent hapten-specific DTH. Thus, the mechanism for regulating the latter may differ significantly from that for CML.

In Vivo Generation of Hapten-Specific Killer T Cells without Elimination of Suppressor Cells

Abstract We have provided evidence to demonstrate that hapten-specific killer cells can be generated in vivo toward hapten-conjugated syngeneic splenic cells. The critical aspect is to provide an auxiliary cellular antigenic stimulus in addition to the hapten-conjugated syngeneic cells. In our experiments this stimulus was CBA/J splenic cells that possess Mls disparate but H-2 compatible antigens with C3H/HeN hosts. The Mls antigen has been shown by others to activate helper T cells in vitro to synthesize KAF, a signal that prekiller cells require besides target antigen. Killer cells (shown to be T cells) as well as helper T cells were found to be derived from the C3H host. Induction in the host animal of partial tolerance to the auxiliary cells possessing Mls antigen abrogated the response. This system, besides providing a better understanding of the control mechanisms involved in the development of T killer cells in vivo, points to a way in which the latter may be generated toward haptenic moieties on syngeneic cells without the necessity for eliminating suppressor cells. The latter principle may prove highly useful in certain clinical situations.

Resurgence of killing and in vivo protection mediated by lymphocytes cultured from lymph nodes draining Moloney sarcomas.

We have previously documented the development and subsequent disappearance of cytolytic activity mediated by lymphocytes from lymph nodes draining Moloney sarcomas destined either to regress or grow progressively. We now report that these tumour-draining lymphnode cells (LNC) that were no longer cytotoxic, spontaneously regenerated peak levels of killing after culture in vitro for 4 days in the absence of exogenous tumour antigen. Cytolytic activity, which was antigenically specific, was mediated by T lymphocytes. Resurgence of cytolytic activity in vitro was accompanied by proliferative changes (DNA synthesis, blast transformation, cell division) which peaked on the 3rd day of culture. Although normal, nonimmune LNC underwent quantitatively similar proliferative changes in culture, the killing that developed was weak and antigenically nonspecific. Transfer of cultured, tumour-draining LNC to immunologically compromised, syngeneic mice conferred complete protection from Moloney sarcoma progression. Adoptive transfer could be delayed for 6 days after tumour induction without loss of protection. These results suggest that there exists in Moloney sarcoma-bearing mice a mechanism that limits the differentiation of pre-killer cells into cytolytically active T lymphocytes, and that such inhibition is eliminated when LNC are explanted into culture.

Influence of killer assisting factor (KAF) on generation of cytotoxic T cells

Generation of killer T cells in murine mixed lymphocyte culture required that prekiller cells be presented with both a killer assisting factor(s) (KAF) and certain cellular alloantigens (presumably H-2K or H-2D) early in the sensitization process. Late addition of either KAF or alloantigen-bearing cells to pre-killer cell cultures decreased the levels of cytotoxicity seen by the fifth day of incubation. In both cases this decrease reflected a delay in the development of cytotoxicity. Periodic removal of KAF from mixed lymphocyte cultures showed that maximum cytotoxicity was obtained when KAF was present along with the alloantigen for the full 5-day sensitization period. However, a significant cytotoxicity could be detected when contact with KAF, in the presence of alloantigen, was allowed for as few as 48 hr of culture. These observations suggest that both alloantigen and KAF must be experienced together to initiate cell mediated lympholysis (CML) development and that full CML development requires the continuous presence of KAF thereafter.

Evidence for a similar or common mechanism for natural killer cell activity and resistance to hemopoietic grafts

AbstractTwo types of host reactivities not requiring immunization in the mouse and not mediated by T lymphocytes were compared: resistance of irradiated and nonirradiated F1 hybrids to accept parental grafts of normal or malignant hemopoietic cells (Hh system), and the natural killer cell activity against mouse lymphomas (NK system). The effects of six independent variables known to influence resistance to marrow grafts were investigated in the NK system using YAC‐1 lymphoma cells as targets. The following properties were shared: (a) maturation during the fourth week of life; (b) low sensitivity to acute total body irradiation; (c) dependence on the integrity of bone marrow as demonstrated by reduced reactivity in 89Sr‐treated mice; (d) suppression by a single injection of rabbit anti‐mouse bone marrow serum; (e) suppression by a single injection of the anti‐macrophage agents silica and i‐carrageenan; and (f) suppression by multiple injections of parental spleen cells into F1 mice. These positive correlations are particularly significant because most of the variables have either opposing or no effect on conventional immunity. F1 mice rendered specifically unresponsive to parental marrow grafts, could retain NK cell activity, and genetically susceptible mice could be rendered hyporeactive in terms of NK cells, indicating that the specificities of YAC‐1 and Hh‐1 incompatible targets were different.It is extremely unlikely that this remarkable parallelism is fortuitous. These results indicate that either a very similar, or more likely a common mechanism is operative in the two cell‐mediated natural reactivities: effector cells in the NK and Hh systems do not bear B or T lymphocyte markers but are nevertheless endowed with “specificity”. They are dependent for generation in vivo (presumably by maturation or by recruitment) on the interaction with nonlymphoid accessory cells not endowed with specificity, capable of also interacting in vitro with Thy‐1‐positive F1 hybrid prekiller cells specific for parental targets. Because of thymus independence in vivo and apparent restriction to target cells of the hemopoietic system, these reactivities should be effective in the regulation of hemopoiesis and surveillance over leukemogenesis.

Splenic T-killer cells can be generated by allogeneic thymic cells in conjunction with assisting factor

CELL-MEDIATED lympholysis (CML) is known to be brought about by thymus-derived (T) killer cells that have acquired reactivity towards antigens borne on target cells. To generate such T killer cells, cooperation between two subsets of T cells within the effector population is required—namely, T helper and T pre-killer cells1–3. Helper T cells are thought to recognise Ia antigens4,5 and/or unique H–2 antigens6, while T pre-killer cells are known to recognise H–2 antigens4,5. Ia antigens are found on B cells as well as on a certain sub-population of T cells7, whereas H–2 antigens have been described on virtually all B and T cells7. Plate has shown that a soluble factor from alloantigen-activated T helper cells assisted T pre-killer cells to differentiate into killer cells8. In this instance, soluble helper factor was generated in separate cultures containing mixtures of allogeneic cells, which was then assayed in a CML system from which T helper cells had been serologically removed. Since most thymic cells are known to lack Ia antigens7, they are conceded to be poor stimulators in both CML and mixed lymphocyte reactions5,9–11. Nevertheless, they possess H–2 antigens and we reasoned that they might still serve to stimulate the generation of killer cells provided an assisting factor was incorporated from an exogenous source. In this way, T killer cells might be generated without at all disturbing the effector cell population by serological deletions. The data presented here support the idea that thymocytes alone are incapable of activating T helper cells, but that in the presence of exogenously produced assisting factor they do trigger development of T killer cells. The data also indicate a simple way by which assisting factors may be studied and by which requirements for CML may be delineated.

Concanavalin A-mediated activation of antigen-primed lymphocytes into secondary cytotoxic lymphocytes.

A secondary specific cytotoxic response is obtained when lymphocytes primed in vivo to a tumor allograft are exposed to Con A in culture. The secondary cytotoxic cells generated are specific to target cells bearing antigens of the primary sensitizing cells and are qualitatively indistinguishable from the response obtained upon secondary antigenic stimulation. The cell-mediated cytotoxicity is independent of concanavalin A (Con A) and is not affected by the Con A-specific inhibitor, alpha-methyl-D-mannose pyranoside. Furthermore, cultures containing a mixture of submitogenic concentrations of Con A and stimulating antigens showed synergy and augmentation of cytotoxic activity. It is suggested that activation of prekiller cells by Con A into CTL may be mediated via the same or similar receptors normally triggered by the stimulating antigens. Functional similarities between ConA and the lymphocyte-defined antigens of the major histocompatibility complex region are discussed.

Regulation of the immune response by subclasses of T lymphocytes. I. Interactions between pre-killer T cells and regulatory T cells obtained from peripheral lymphoid tissues of mice.

The generation of killer cells to alloantigens in vitro depends upon an interaction between two subclasses of peripheral T cells: "pre-killer" T cells and "regulatory" T cells. The pre-killer T cell, found in highest concentrations in peripheral lymph nodes of mice, is sensitive in vivo to the administration of small doses of anti-thymocyte serum (ATS) and is depleted in vitro by treatment with (a) anti-Thy-1.2 + C, (b) low doses (600 r) of x-irradiation. Specificity of pre-killer cells is supported by adsorption of pre-killer activity on appropriate allogeneic monolayers and by the specific absence of this activity from lymphoid tissues of mice rendered tolerant in neonatal life. Although T cells remaining in the spleen 2 days after a small dose of ATS (SPA-T) did not generate killer cells, this subset exerted substantial regulatory effects upon the generation of killer activity by pre-killer T cells. The addition of this population of regulatory T cells to small numbers of lymph node T cells (LN-T) resulted in substantial enhancement in the generation of killer cells from the LN-T pre-killers. The addition of SPA-T to larger numbers of LN-T (which produce strong responses alone), failed to enhance the response, and in some cases resulted in significant suppression which could not easily be accounted for by alteration in cell numbers in culture. Although amplifier activity was relatively radioresistant, "suppressor" activity was sensitive to relatively small doses of irradiation. The significance of this T-T interaction is discussed.