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B Bonavida

Publications and source records attributed to B Bonavida.

At least 127 records · Page 7Linked to original sources
PubMed1987

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity.

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.

AIDS-Related Complex↗
PubMed1987

Regulation of Ia+ reticulum cell sarcoma (RCS) growth in syngeneic SJL/J mice. I. Inhibition of tumor growth by passive administration of L3T4 monoclonal antibody before or after tumor inoculation.

Spontaneously arising reticulum cell sarcoma (RCS) tumors in SJL/J mice stimulate syngeneic host T lymphocytes to proliferate and are dependent on host T cells for maintenance and growth. Tumor-associated Ia antigens have been implicated in the proliferative response both in vivo and in vitro, and the responding T cells are predominantly Lyt-1+2- L3T4+. We hypothesized that elimination or depletion of the responding L3T4 subpopulation in vivo should inhibit growth of transplantable RCS tumors, and continued RCS growth may be dependent on the continued presence of L3T4 cells. This hypothesis was tested experimentally by examining the effect of passive administration of L3T4 monoclonal antibody (mAb) into SJL/J mice either before or at different times after tumor inoculation. The tumor inoculum used killed all mice 15 to 30 days after injection. Administration of a single dose of L3T4 mAb 4 days before tumor inoculation resulted in complete depletion of L3T4 cells and complete inhibition of tumor growth. The antibody-treated mice survived with no sign of tumor growth even after complete recovery of L3T4+ cells. These results demonstrate that initiation of tumor growth is dependent on host L3T4+ cells. Administration of mAb as late as 7 days after tumor inoculation resulted in inhibition of tumor growth, and administration of mAb at day 10 resulted in significant inhibition of tumor growth. Compared with the kinetics of tumor growth in normal control mice, administration of L3T4 after tumor inoculation results in tumor growth arrest. These findings demonstrate that continued tumor growth in vivo is dependent on the presence of L3T4+ cells. In the RCS system, the present studies show that administration of mAb to L3T4+ cells is therapeutic in that it inhibits the induction of tumor growth, and it also prevents tumor growth in tumor-bearing animals.

Animals↗
PubMed1987

Studies on the mechanism of natural killer cell-mediated cytotoxicity. VII. functional comparison of human natural killer cytotoxic factors with recombinant lymphotoxin and tumor necrosis factor.

The present study was undertaken to evaluate the possible contribution of other cytokines to the lytic activity of NKCF-containing supernatants. We compared some of the functional properties of human NKCF and purified recombinant human rLT and rTNF. It was found that the target cell specificity of rLT was quite different from NKCF in that rLT was neither species specific nor NK specific. Furthermore, antibodies against rLT did not affect the lytic activity of NKCF. These results demonstrate that LT does not significantly contribute to the lytic activity mediated by NKCF. The target specificity of rTNF was found to be related to that of NKCF with the exception of one NK-resistant cell line that was lysed by rTNF in a 20-hr 51Cr-release assay. However, rTNF was not toxic to any of the target cells tested as assessed by trypan blue exclusion in a 20-hr assay unless the targets were labeled with 51Cr. In contrast, NKCF did kill target cells as detected by trypan blue exclusion that were not labeled with 51Cr. Further analysis of this mechanistic difference in the lytic activity of rTNF and NKCF revealed that rTNF in combination with either cycloheximide or mitomycin C but not IFN-gamma could lyse unlabeled U937 target cells. In addition, pretreatment of U937 target cells with nonradioactive Na2CrO4 at concentrations equivalent to that used to 51Cr-labeled cells resulted in their susceptibility to lysis by rTNF as assessed by trypan blue exclusion. These findings suggest that lysis of several susceptible target cells in 20 hr by rTNF requires the presence of additional agents that may be sublethally toxic and/or inhibitory to macromolecular synthesis. Antibody inhibition studies revealed that anti-TNF mediated from partial to complete inhibition of lysis of U937 by unfractionated supernatants containing NKCF. However, fractionation of such supernatants on chromatofocusing columns yielded two distinct peaks of activity eluting in the pH range of 5 to 6 and 7 to 8. Anti-TNF could inhibit the acidic form of NKCF but not the neutral form. It is concluded that NKCF activity is mediated in part by TNF or an antigenically related molecule as well as some other distinct factor(s). The lack of consistent inhibition of NK CMC by anti-TNF suggests that TNF alone is not sufficient to mediate NK activity, or else it is inaccessible to the added antibody.

Antibodies, Monoclonal↗
PubMed1987

Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF).

Growth and differentiation of B cells into Ig-secreting plasma cells is regulated by both T cells and macrophages and/or their secreted factors. Although the regulatory role of various cell-derived factors has been examined, the involvement of the macrophage-derived factor, TNF, in human B cell growth and differentiation has not yet been investigated. In the present study we examine the role of rTNF in polyclonal B cell response of human PBL induced by PWM. The addition of rTNF at the initiation of the culture resulted in the dose-dependent inhibition of the generation of both IgG and IgM PFC. Inhibition of PFC development followed the same dose response as rTNF-mediated cytotoxicity against a TNF-sensitive tumor target. The mechanism of rTNF-mediated suppression was examined in different experimental systems. Recombinant TNF did not affect the viability or proliferation of either the T cell or B cell subpopulations, suggesting that TNF does not mediate its suppressive effect by cytotoxic mechanisms. Kinetic studies in which rTNF was added at different times after initiation of culture indicated that inhibition can be observed as late as 4 days of culture and suggested that TNF acts at a late phase of the growth and differentiation pathway of B cells. In further studies we examined the cellular level of TNF-mediated suppression. The addition of rTNF to supernatants containing helper factors and enriched B cells resulted in no inhibition, suggesting that TNF does not act at the B cell level. This was confirmed by demonstrating that rTNF does not inhibit spontaneous PFC development by the CESS B cell line. The effect of TNF on T cell subpopulations was examined by using normal or irradiated T cells, which inactivate suppressor cells. Addition of rTNF to B cells combined with either T cell population suppressed both IgG and IgM PFC development, indicating that the target cell for suppression is the T helper cell but not ruling out an effect on macrophages or the T suppressor cells. Combined, the observed results demonstrate that rTNF suppresses PWM-induced B cell differentiation without affecting B cell proliferation. TNF appears to mediate the suppression by acting directly on T helper cells or else by regulating the production of factors controlling T cell activation and lymphokine secretion.

Antibody Formation↗
PubMed1987

Detection of a transforming gene in spontaneous reticulum cell sarcoma of SJL/J mice: genetically linked and host-dependent neoplasia.

Spontaneous reticulum cell sarcoma (RCS) tumor induction occurs in 90% of SJL/J mice of 8-13 months of age. Tumor induction and growth has been shown to be under the influence of both H-2 and non-H-2 genes as well as the presence of an intact host T-cell system. We postulated that cellular oncogenes may play a role in the induction, growth, and characteristics of RCS. DNA-mediated gene transfer protocols were adopted to investigate the presence of transforming genes in DNA from RCS of SJL/J mice. High molecular weight DNA was isolated from these tumors as well as from brains and livers of control tumor-free SJL/J mice and transfected into NIH-3T3 mouse and F2408 rat fibroblast cell lines. Foci of transformed cells with a peculiar round morphology were scored in both rat and mouse cultures given tumor DNA, but not in those receiving DNA from normal tissues. DNA from first-cycle transformants was transfected in further cycles of transfection, giving rise to foci with similar morphological appearances and growth properties. These experiments suggest that a transforming gene, present in RCS spontaneous tumors, is involved in the malignant conversion of the transfected normal fibroblasts. The implication of these results with respect to the induction and growth properties of RCS is discussed.

Animals↗
PubMed1987

Lysis of natural killer-sensitive and -resistant tumor cells by natural killer cytotoxic factors (NKCF)-containing liposomes.

The lethal hit stage in NK cell-mediated lysis requires a complex series of events involving the release of NKCF, subsequent binding of these factors to the target cell, and susceptibility of the target cell to lysis by NKCF. Binding of NKCF alone is not sufficient because a number of tumor cells are able to bind NKCF without being lysed, suggesting the need for an additional processing step active on susceptible target cells. In the present study, we show that the interaction with liposome-incorporated NKCF renders NK resistant target cells sensitive to NKCF-mediated lysis. These results suggest that NKCF may mediate their cytotoxic effects through internalization of these factors into the cytosol.

Animals↗
PubMed1986

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding.

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.

Animals↗
PubMed1986

Mechanism of NK activation by OK-432 (Streptococcus pyogenes). I. Spontaneous release of NKCF and augmentation of NKCF production following stimulation with NK target cells.

The biological response modifier OK-432 (Picibanil) (manufactured in Japan) is produced by lyophilization of cultures of the low virulent Su strain of group A Streptococcus pyogenes of human origin. This preparation has been shown to have multiple effects on the immune system and has been used as an anti-cancer therapeutic agent in man. It has been shown that OK-432 augments the cytotoxic activity of human natural killer (NK) cells. We have proposed that natural killer cytotoxic factors (NKCF) derived from NK cells play a role in the mechanism of NK cell-mediated cytotoxicity (CMC). The present study investigates the underlying mechanism of the OK-432-mediated enhancement of NK activity by determining whether OK-432 has an effect on the induction and activity of NKCF produced by NK cells. Treatment of peripheral blood lymphocytes (PBL) with OK-432 for 20 hr and wash resulted in significant augmentation of NK CMC and this enhancement was dependent on the concentration of OK-432 used. Coculture of the OK-432-treated PBL with U937 resulted in a several-fold enhanced production of NKCF in the supernatant. The NKCF produced were similar to those produced by untreated effector cells in that they had the same NK target specificity for lysis. The time kinetics of stimulation of PBL with OK-432 for optimal production of NKCF was found to be 8-12 hr. It was also observed that culture of OK-432-treated PBL in the absence of stimulator cells spontaneously release significant amounts of NKCF into the supernatant. The supernatant containing NKCF was tested for interleukin 2 (IL-2) activity using an IL-2-dependent HT-2 line. It was found that there was no direct correlation between the levels of NKCF and IL-2 activity. The results of this study demonstrate that OK-432 stimulates NK cells to produce NKCF in the presence or absence of stimulator cells. The optimum concentration of OK-432-induced augmentation of NK CMC paralleled that seen for optimum NKCF production, suggesting that one mode of action of OK432 is to enhance NKCF production in a manner reminiscent of IFN and IL-2. The results also point out that OK-432 acts by a mechanism independent of the action of IL-2.

Biological Products↗
PubMed1986

Studies on the lethal hit stage of natural killer cell-mediated cytotoxicity. I. Both phorbol ester and ionophore are required for release of natural killer cytotoxic factors (NKCF), suggesting a role for protein kinase C activity.

Previous studies in our laboratory on the natural killer (NK) lytic mechanism demonstrated that following interaction of target cell with effector cell, the effector cell releases NK cytotoxic factors (NKCF) that can then bind to and lyse the target cell. This study investigates the mechanism by which the target cell signals the effector cell to release NKCF. Studies on other cell systems with secretory functions have indicated that receptor-induced transmembrane signaling leads to the metabolism of phosphatidylinositol and activation of protein kinase C (PKC) by increased cytosolic Ca++ and diacylglycerol (DAG). We tested the hypothesis that a similar sequence of activation events occurs in human NK cells by examining the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the calcium ionophores A23187 and ionomycin in their ability to induce release of NKCF. The amount of NKCF released was determined in a 20-hr 51Cr release assay against an NK-sensitive target cell. A23187, ionomycin, or TPA alone did not induce release of NKCF. However, ionophores (200 mM) in conjunction with TPA (20 ng/ml) induced release of NKCF. Several properties of the induced NKCF by TPA and ionophores were concordant with those of the NK cell-mediated cytotoxicity (CMC) reaction. The kinetics of release were faster (less than 1 hr) than when either Con A or target cells were used to stimulate NKCF. Only NK-sensitive target cells were killed by NKCF. Pretreatment of effector cells with interferon enhanced release of NKCF from effector cells. Several lines of evidence suggested that the pathway of activation takes place through phosphatidyl inositol metabolism. Activation of PKC was indicated because TPA and A23187 enhanced protein phosphorylation in the LGL-enriched fraction. Experiments that made use of oleoyl acetyl glycerol, a synthetic DAG, showed release of NKCF in the absence of A23187 but was augmented by the ionophore. The above studies suggest that NKCF is released from NK effector cells within a period of time consistent with NK CMC, and the release of NKCF results either directly or indirectly from protein phosphorylation by PKC.

Calcium↗
PubMed1986

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. I. Defective trigger on NK cells for NKCF production by target cells, and partial restoration by IL 2.

Peripheral blood from patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) exhibits poor NK activity in the 51Cr-release assay. The present studies were undertaken to investigate the mechanism underlying the observed defective NK cytotoxic activity. On the basis of our studies on the mechanism of natural killer cell-mediated cytotoxicity (NKCMC), a defective NK cell can result from lack or decreased frequency of effector cells, inability to recognize and bind the target cell, failure to be activated for the release of NK cytotoxic factors (NKCF), and/or failure to synthesize or secrete NKCF. Each of these various possibilities was examined. Single cell analysis revealed that the frequency of NK cells was comparable to controls, and although the NK cells bind to the NK-sensitive target, the bound target is not lysed. These results suggested that the defect in NK cells was not due to depletion of NK cells or to a defect in recognition structures, but that it was located at the postrecognition event. We previously demonstrated that after binding to target, the NK cell is stimulated to release NKCF in the supernatants and NKCF lyse specifically NK-sensitive targets. Accordingly, we investigated the activation of NK cells from AIDS and ARC patients for release of NKCF. After coculture with the stimulator cell, the patients' NK cells failed to release active NKCF in the supernatant. However, the cells released NKCF after stimulation with the lectin Con A or a mixture of TPA and ionophore, albeit to a lesser extent than controls. These results suggested that AIDS and ARC NK cells are defective in the trigger involved in release of NKCF. Further studies were done to investigate whether the immunomodulator IL 2 can restore the functional activity of the defective NK cells. Treatment with IL 2 resulted in augmented NK cytolytic activity, but did not reach control levels of activated cells from normal controls. Furthermore, the patients' IL 2-treated cells recover partially the ability to be stimulated by NK cells and to release NKCF. These results suggest that the trigger for NKCF production and the cytolytic function of the patients' NK cells are regulated by IL 2. By delineating the stage at which the AIDS and ARC NK cells are defective, it is now possible to monitor their recovery and to investigate the effect of various biologic response modifiers in restoring NK activity.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Mapping of SJL/J reticulum cell sarcoma tumor-associated Ia antigens by T cell hybridomas: characterization of tumor-specific and shared epitopes detected on IE+ allogeneic cells.

Previous studies have suggested that reticulum cell sarcoma (RCS) tumor cells of SJL/J (IA + IE-) mice express neospecificities that are related to antigenic specificities characteristic of IE+ allogeneic cells. These neospecificities have also been suggested to play a role in the strong syngeneic antitumor proliferative response as well as in regulating RCS growth in vivo. The present studies characterize four RCS tumor-specific T cell hybridoma clones prepared from the fusion of BW5147 thymoma with T cells derived from lymph nodes of tumor-bearing mice. Upon stimulation, these hybridomas secrete IL 2 in the supernatant. Two hybridomas responded to RCS to IE+k and to IE+d allogeneic cells, respectively, and the other two hybridomas were tumor specific. The specificity of these hybridomas was assessed by response to both spontaneous and transplantable RCS lines and failure to stimulate a response by either normal or LPS-induced B cell blasts from the host SJL/J cells. The epitopes recognized by the T cell hybridomas were examined by the ability of several monoclonal antibodies to inhibit the IL 2-induced response by the T cell hybridomas. Antibodies directed against the IABs polypeptide of the IA hybrid molecule blocked the antitumor response by all four hybridomas. However, the response to allogeneic IE+ cells was not blocked by anti-IAs antibody but was blocked by antibodies directed against either the IAk,d or IEk,d hybrid molecules or the corresponding alpha- or beta-chains. The response to both RCS and allogeneic cells was blocked by monoclonal antibodies directed against L3T4 antigens on the T cells. Based on the exquisite specificity of the T cell receptors, the results here demonstrate that RCS tumor cells express on their surface both tumor-specific I-A-associated epitopes and Ia-associated antigenic specificities that are shared with IE+ allogeneic cells. The present studies of adapting T cell hybridomas and blocking antibodies proved useful to characterize and map distinct tumor-associated epitopes on the surface of tumor cells. These findings, when combined with structural studies, should help unravel the molecular complexity of tumor-associated antigens.

Animals↗
PubMed1986

Effect of altered membrane structure on NK cell-mediated cytotoxicity. II. Conversion of NK-resistant tumor cells into NK-sensitive targets upon fusion with liposomes containing NK-sensitive membranes.

There is a large body of evidence that supports the notion that NK cells exert important immune surveillance functions in vivo, against a variety of virus-infected and neoplastic cells. However, certain targets are not susceptible to lysis by NK cells. The exact mechanism by which resistance or sensitivity is conferred on target cells is not known. We investigated whether the selectivity to NK lysis is a property of the membrane of the target cell. This was examined by the application of a recently developed method which is aimed at changing the membrane structure of the target cell by cell-liposome fusion. Our studies demonstrate that NK-resistant tumor cells acquired sensitivity to lysis by NK cells after fusion with reconstituted vesicles which contained membrane components derived from NK-sensitive target cells. The fusion required the presence of Sendai virus envelope glycoproteins and exogenous lipids (soybean lecithin and cholesterol) for maximal efficiency. This finding was demonstrated in both the human system (with U937 and Raji as NK-sensitive and -resistant cell lines, respectively) and the rat/murine system (with YAC-1 as NK-sensitive target and P815 and YAC-asc as NK-resistant targets). Both the 51Cr-release assay and the single cell assay showed lysis of the modified target cells in a 3-hr incubation period. The magnitude of the cytotoxic activity was found to depend on the concentration of reconstituted vesicles used in the fusion step. The effect seen was specific because target cells were not lysed when fused with vesicles which contained membrane constituents derived from either NK-resistant targets or NK-sensitive targets from another species (human vs mouse). The resistance of modified target cells to lysis by xenogeneic NK cells was not due to failure of membrane fusion, as detected by immunofluorescence, or to failure to form conjugates. These results demonstrate the feasibility of converting a resistant NK target to a sensitive target by cell-liposome fusion. Furthermore, the data indicate that susceptibility to lysis by NK cells is a property of the membrane composition of the target cell. The significance of these findings is discussed.

Animals↗
PubMed1986

Role of natural killer cytotoxic factors in the mechanism of target-cell killing by natural killer cells.

Studies on the mechanism of cell-mediated cytotoxicity (CMC) have suggested a stimulus-secretion model and implicated a role of soluble cytotoxic mediators. Our studies in the natural killer (NK) system provide several lines of evidence for the involvement of natural killer cytotoxic factors (NKCF) in NK CMC and led to the development of a model for the NK lytic mechanism. This model delineates several interactions between NK cells and targets that are deemed necessary to achieve target-cell lysis. The first stage is the interaction of the effector with the target cell, resulting in contact and adhesion. This is presumably mediated by NK recognition structures and target-cell structures. Following binding, the target cell stimulates the NK cell to release NKCF. This step is functionally distinct from the initial effector-target binding. The trigger mechanism for release of NKCF appears to be dependent on protein kinase C. The released NKCF binds to NKCF binding sites on the target cell followed by "processing" or "internalization" and, ultimately, resulting in cell death. This model has been shown to be useful in investigating the mechanism of defective NK activity in certain disease states. Biochemical analysis and comparative studies suggest that NKCF is a distinct molecule from other cytotoxins studied to date. The studies in the NK CMC system supporting a role of cytotoxic mediators also suggest a possible role for cytotoxic factors in other cytotoxic systems. Furthermore, the selective susceptibility to lysis of tumor or infected cells by NKCF suggests a possible role of their effectiveness in in vivo therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1986

Analysis of lymphocyte-target conjugates by flow cytometry. I. Discrimination between killer and non-killer lymphocytes bound to targets and sorting of conjugates containing one or multiple lymphocytes.

The use of flow cytometric analysis and sorting techniques for the enumeration and purification of lymphocyte-target conjugates was investigated. Murine cytotoxic T-lymphocytes (CTL) with killer effector function were identified and quantitated during a 3-hour cell-mediated cytotoxicity reaction using multiparameter analysis. Resolution of conjugates containing single and multiple lymphocytes was achieved by two-color fluorescence, and individual conjugate subpopulations were subsequently sorted for further analysis. To measure total and cytotoxic conjugate frequencies, CTL were labelled with FITC-conjugated Thy 1.2 antibody and dead target cells were stained with propidium iodide (PI). Size difference between the CTL and P815 tumor target cells, as measured by Coulter volume and axial light loss, facilitated detection of conjugates which were identified as both large and Thy 1.2-positive. Conjugates containing dead target cells possessed red fluorescence due to PI uptake. The frequency of conjugates containing cytotoxic activity increased with time during the cytotoxicity period and correlated with frequencies obtained in single-cell assays. Analysis of the distribution of single and multiple lymphocyte-bound conjugates was done by co-centrifugation of Hoechst-stained CTL and FITC-labeled P815 target cells. Analysis by two-color fluorescence effectively resolved conjugate populations containing different numbers of CTL and allowed their purification by cell sorting. The purity of the separate populations was confirmed by fluorescence microscopic inspection. The results of these studies demonstrate that flow cytometry can resolve target-bound and free CTL, measure cytolytic efficiency and specifically sort out cytometrically defined subgroups within the effector cell population.

Animals↗