Quantitative analysis of the 51Cr release cytotoxicity assay for cytotoxic lymphocytes

Using ⁵¹Cr-labelled P-815 mastocytoma cells as target cells and CS7BL/6 spleen cells sensitized against DBA/2 antigens as effector cells, it is shown that the variation in the observed specific ⁵¹Cr release over a broad range of experimental conditions can be explained on the basis of a simple physical model of the interaction process. The model assumes that a target cell can be destroyed only after contact with an effector cell, contact takes place on a random basis, one contact is sufficient, and that one effector cell can kill several targets with unchanged efficiency. The fraction of target cells destroyed (f) depends only on the incubation time (t), the number of effector cells (n) and a constant interaction probability (δ). Thus f = 1 ? e^?nδt. However, the experimental measurement, the fraction of ⁵¹Cr specifically released into the supernatant during the assay, may not be the same as the fraction of target cells destroyed because it takes considerable time for the releasable ⁵¹Cr to be released from a damaged target cell. This can be overcome experimentally by following the standard 37 °C incubation with a further incubation at 45 °C during which there are no new lytic events but all previously damaged target cells release the remainder of their releasable ⁵¹Cr. The model enables one to obtain accurate measurements of relative effector cell frequency over a broad range of experimental conditions.     

T-rosettes in hemodialysis patients and renal allograft recipients

Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by ³H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release ⁵¹Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.     

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 (⁵¹Cr) to label target cells. These targets are then exposed to effector cells and the release of ⁵¹Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of ⁵¹Cr. As effector cells, we used an activated autoreactive clonal population of CD8⁺ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with ⁵¹Cr labeled target NIT cells for 16 hours, release of ⁵¹Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO₂, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.     

Measurement of CTL-induced cytotoxicity: The caspase 3 assay

Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the ⁵¹Cr release assay. However, measurement of target cell lysis by ⁵¹Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the ⁵¹Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.     

T-lymphocyte mediated lysis of tumor cells in the presence of alloantiserum.

The blocking effects of tumor alloantiserum (AS) on the process of T-lymphocyte mediated cytolysis of target cells was investigated with the scanning electron microscope by analyzing the frequency of lymphocyte-target cell interactions and the respective changes in target cell morphology at various time intervals of the cytolytic process. Our results demonstrate that in the presence of AS the frequencies of lymphocyte-target cell conjugates in which the target cells were undergoing active blebbing correlated with the delayed kinetics of ⁵¹Cr release. Our data also show that the AS did not interfere significantly with the binding of lymphocytes to target cells, but delayed the appearance of surface blebbing of the target cells. Thus, effector lymphocytes required a prolonged time of continuous interaction with the target cells in order to exert their cytolytic effects in the presence of AS. This conclusion was further confirmed by experiments in which lymphocyte-target cell interactions were interrupted by the addition of EDTA to the culture medium.     

THE MECHANISM OF LYMPHOCYTE-MEDIATED CYTOTOXICITY

1. Three classes of cytotoxic lymphocyte are discussed: thymus-derived T cells, antibody-dependent K cells and NK (natural killer) cells. Each of these cytotoxic lymphocytes has receptors allowing the formation of adhesions (contact) with a target cell (the cell to be killed). The type of receptor and the corresponding ligand on the target cell is different in each class. Cytotoxic T cells (and probably NK cells) react with a target cell antigen, in a manner rather like antibody-antigen reactions (although not involving classical serum antibody). K cells have a receptor for the Fc part of immunoglobulin (IgG) and hence can make contact with antibody-coated target cells. 2. It seems likely that all three classes of cytotoxic lymphocyte have a similar basic mechanism of killing, which is different from the membrane leakage occurring in complement-mediated lysis. Much more information is available on cytotoxic T cells than on the other types of cell. 3. Cytotoxic T cell killing can be divided into two phases. A reversible phase in which the T cell is in contact with the target cell, but causes no apparent damage. This phase can vary from a few minutes up to several hours, when a single T cell interacts with a single target cell. If the T cell detaches or is inactivated the target cell survives. The second phase is irreversible, once the lethal event has occurred, and the target cell will progress to eventual lysis in the absence of the Tc cells. 4. The lethal event initiates a period of zeiosis (membrane blebbing) in the target cell, which is accompanied by increased effiux of ⁸⁶rubidium. Cell lysis occurs at a variable time after the initiation of zeiosis, when the soluble contents of the cytoplasm burst out of the target cell. The fact that both these phases are of variable length leads to the accumulation of cytoplasmic markers (such as ⁵¹chromium) in the medium in an approximately linear fashion. 5. The nature of the lethal event is unknown, but it is suggested that it involves changes inside the target cell rather than at the target cell membrane. Remarkable long projections from the T cell (and also seen from K cells and NK cells), apparently arising as a result of the receptor-ligand interaction, may be involved in the delivery of the lethal event.     

Contrasting effects of alloantiserum and xenoantiserum on cell-mediated lysis of tumor cells

The effect of anti-EL-4 serum on antibody-dependent cytotoxicity (ADCC) and cell-mediated cytotoxicity (CMC) was studied in allogeneic and xenogeneic systems. Inbred strains of BALB/c mice and Lewis rats were immunized with EL-4 tumor cells. Using microcytotoxic assays of ⁵¹Cr release from labeled EL-4 cells, complement-dependent cytolysis, ADCC, and CMC were determined. Complement-dependent cytolysis was observed in both systems. Although ADCC was demonstrated in both systems, the kinetics of cytolysis were different. Xenoantisera and alloantisera had opposite effects on CMC. Incubation of EL-4 target cells with BALB/c anti-EL-4 serum resulted in inhibition of CMC by immune BALB/c spleen cells. In contrast, treatment of EL-4 target cells with Lewis anti-EL-4 serum potentiated the CMC of immune Lewis spleen cells. It is thought that differences in the strength of response, antibody characteristics, and effector cells may determine the degree of inhibition or potentiation observed in these systems.     

Rapid kinetics of lysis in human natural cell-mediated cytotoxicity: Some implications

The entire lytic process of natural cell-mediated cytotoxicity against sensitive target cells can occur rapidly, within minutes. This was demonstrated by ⁵¹chromium release and in single-cell assays. At the cellular level, most of the target cell lysis occurred within 15–30 min after binding to effector cells. The enriched natural killer cell subpopulation of lymphocytes obtained by Percoll density gradient centrifugation (containing >70% large granular lymphocytes (LGL)) was the most rapidly lytic population by ⁵¹chromium release. However, in the single-cell assay, the rate of lysis of bound target cells was quite similar for the LGL-enriched effector subpopulation and the higher density subpopulation of effector cells recognized previously. Both the light and dense effector cells contained similar numbers of target binding cells. Therefore, that the light subpopulation effected lysis more rapidly and to a greater extent than the dense subpopulation suggested that the low-density effector cells probably recycled more rapidly than those of higher density. This was corroborated by the finding that when conjugates were formed at 29 °C for the single-cell assay, a significant number of dead unconjugated targets could be observed only on the slides made with the LGL-enriched effector cells but not on those made with dense effector cell. Lysis continued to increase in the chromium-release assay probably because of recycling, recruitment, and/or heterogeneity of the effector cells, and/or because of heterogeneity or delayed death of the target cells.     

The efficiency and linearity of the radiochromium release assay for cell-mediated cytotoxicity.

The release of radiochromium from EL-4 cells lysed by anti EL-4 peritoneal lymphocytes is a first order decay process with respect to time. The rate of ⁵¹Cr release from damaged cells was independent of the effector: target ratio but was slower from pelleted cells than cells in suspension. We conclude that under normal assay conditions the fraction of chromium released is nearly proportional to the number of cells damaged but find that the proportionality constant is influenced by assay conditions. There is a lag time of 23 ± 5 min before ⁵¹Cr is rapidly released from the cells.     

Heterogeneity of human natural killer cell populations.

Natural killing (NK) in human donors was determined by the ability of peripheral blood subpopulations to lyse the myeloid target, K562, in a 2 to 4 hr ⁵¹Cr release assay. The most active cell was a non-T cell which passed through nylon columns (representing 10 to 25% of column passed cells). A second column passed cell population, with characteristics of T lymphocytes (75 to 90% of column passed cells), was also capable of mediating natural killing. Non-T cells which were retained by the nylon columns (45 to 55% of adherent cells) lacked NK activity. However, nylon adherent T cells (45 to 55% of adherent cells) were consistently active in NK assays, illustrating an important subset of NK effector cell often overlooked. Both column passed and adherent T cells were further separated according to their ability to bind IgG or IgM immune complexes, showing that those mediating NK have receptors for IgG (Tγ+) but not for IgM (Tμ+).     

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Lysis of virus-infected target cells by antibody-dependent cellular cytotoxicity. I. General requirements of the reaction and temporal relationship between lethal hits and cytolysis.

The effect of various physical and chemical parameters on the cytotoxic reaction was studied in a ⁵¹Cr-release assay in order to analyze the mechanism by which human blood mononuclear cells (MC) damage antibody-sensitized target cells infected with herpes simplex virus. Centrifugation of the target cell-MC mixture consistently increased the velocity of the reaction. In addition, uncentrifuged target cell-MC cultures showed a sigmoidal kinetic curve of ⁵¹Cr release with an initial lag phase of at least 10 min, whereas ⁵¹Cr release in centrifuged cultures followed a linear pattern with time without an initial lag. These findings indicate that direct contact between target and effector cells is necessary for cytotoxicity to occur. The reaction as a whole was temperature dependent, proceeding well at 37 °C and not at all at 4 °C. Incubation of the MC at 46 °C for 10 min abolished their cytotoxic potential without affecting their viability; similar heating of the target cells did not affect their background isotope release or sensitivity to the lytic process. Heating target cell-MC mixtures at 46 °C for 10 min thus provided a tool by which the temporal relationship between the mounting of “lethal hits” and specific isotope release, or cell lysis, could be studied. Using this technique, we observed virtually simultaneous occurrence of lethal hits and cell lysis, measured at various intervals between 10 and 360 min postincubation. Likewise, we were unable to demonstrate a transient period of increased osmotic fragility in target cells after contact with MC but before actual cell lysis. Taken together, these findings imply either that cell lysis, as indicated by ⁵¹Cr release, results from a sudden nonosmotic injury to the target cell membrane or, alternatively, osmotic damage leading to ⁵¹Cr release occurs too rapidly to be detected by the methods employed in this study. These findings imply either a qualitative or a quantitative difference between antibody-dependent cellular cytotoxicity (ADCC) mediated by K cells and cytotoxicity mediated by sensitized T cells.The cytotoxic reaction was completely inhibited by 10 mM EDTA and did not occur in a Ca²⁺- and Mg²⁺-free medium. Neither Ca²⁺ nor Mg²⁺ alone produced as much cytotoxicity as the two cations in tandem; in addition, when added to the culture medium in suboptimal amounts, the two cations were either additive or synergistic. These observations suggest that both cations are necessary in ADCC and also that there may be separate Ca²⁺- and Mg²⁺-dependent events in the lytic pathway.     

Announcements

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Contact regions of cytotoxic T lymphocyte-target cell conjugates.

The binding of cytotoxic T lymphocytes to target cells was studied by ultrastructural and tracer techniques. It was found that binding was achieved through interaction of the microvilli of both cells and that only a relatively small proportion of the cell surface was involved. Short points of contact, averaging 1500 Å in length, were the main form of junction. Periodic substructures were observed in some of the contact points. The transfer of cytoplasmic content from effector to target cell and vice versa was investigated, but no fluorescein or ⁵¹Cr-labeled components were transferred during the interaction. Examination of cell organelle localization during the interaction revealed that microfilaments were the only cellular components which localized at the contact area; the well-developed Golgi apparatus of the cytotoxic lymphocytes was randomly distributed.     

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder

Summary Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8⁺/CD4^–). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.     

Chicken Harder's gland: evidence for a relatively pure bursa-dependent lymphoid cell population

Theophylline, caffeine, and dibutyryl cAMP, agents that elevate intracellular levels of cyclic AMP, were found to inhibit cytotoxin elaboration by PHA-stimulated human lymphocytes. Cytotoxins in diluted supernatants from 3-day lymphocyte cultures were assayed by ⁵¹Cr release from L cells. Addition of the agents to the lymphocyte cultures inhibited cytotoxin elaboration 70–90% at 10^?3M and 20–50% at 10^?5M. In addition, it was found that the inhibition is reversible and occurs at a step other than the initial mitogen triggering.The inhibition of cytotoxin elaboration by these agents correlates strikingly with their inhibition of lymphocyte-mediated target cell lysis. The results are consistent with the hypothesis that cytotoxin is the mediator of target cell killing by lymphocytes.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

Differential 51Cr uptake of human peripheral lymphocytes separated by density gradient electrophoresis

Human peripheral lymphocytes were labeled with ⁵¹Cr before or after separation by preparative density gradient electrophoresis. In both cases, wide variations in the distribution of ⁵¹Cr in the electrophoresed cells was observed. In general, there was significantly more ⁵¹Cr per cell in the high mobility fractions. These results suggest caution in the interpretation of cytotoxic assays where ⁵¹Cr-labeled lymphocytes are used as target cells and prompt further studies by different separation methods.