Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL- TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. II. Electrolyte permeability increase in the target cell membrane concomitant with programming for lysis.

In a previous study of the mechanism of specific target cell lysis by alloimmune cytolytic T lymphocytes (CTL), we established that the target cell becomes irreversibly programmed to lyse within a few minutes after contact with the CTL. We here show that at each point in time, the level of specific release of the potassium analog, 86Rb equals the percentage of target cells which have been programmed to lyse. It is also shown that specific release of 86Rb is more rapid than that of a small metabolite of similar weight, 14C-nicotinamide, which in turn is specifically released more rapidly than 51Cr. Thus, an electrolyte-permeable lesion is produced in the target cell membrane within minutes of contact with the CTL. Since measurements of 86Rb release, unlike measurements of programming for lysis, do not involve exposure of the cells to EDTA and vigorous shearing forces, the present observations corroborate and extend, by an independent and gentler method, our previous conclusion that the CTL effects crucial and irreversible changes in the target cell within minutes after contact. The present results are consistent with the possibility that the first, and perhaps the only damage administered directly by the CTL is a membrane lesion permeable to electrolytes and possibly to small molecules.     

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.     

Scanning electron microscopic study of natural killer cell-mediated cytotoxicity

The interaction between human natural killer (NK) cells and NK-susceptible target cells, as well as the mechanism involved in target cell lysis, were studied with scanning electron microscopy (SEM). Low density human peripheral blood lymphocytes, highly enriched with large granular lymphocytes (LGL), were used as effector cells, and K562-cells were used as NK-susceptible target cells. The surface features of LGL/NK cells were examined under SEM. In the area of interaction, NK/target-cell conjugates showed microvilli and/or filipodia, and extensive areas of intercellular contact. In addition, the effector cells in some NK/target-cell conjugates were polarized toward the target cell. Changes in target cell surface features included loss of microvilli, large surface blebs and the appearance of small pore-like lesions on the cell membrane. Our findings show that target cell lysis occurred by apoptosis and plasma membrane lesions analogous to those seen during complement-mediated cytotoxicity.     

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.     

Target cell death triggered by cytotoxic T lymphocytes: a target cell mutant distinguishes passive pore formation and active cell suicide mechanisms.

The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths.     

Induction of target cell apoptosis by channel catfish cytotoxic cells.

This study examines cytotoxic mechanisms used by channel catfish peripheral blood-derived effector cells. Transmission electron microscopy (TEM), coupled with [(3)H]thymidine DNA fragmentation (JAM) and terminal deoxynucleotidyl nick-end labeling (TUNEL) assays, provided the first evidence that catfish peripheral blood cytotoxic effectors killed allogeneic targets via an apoptotic pathway. TEM demonstrated that the effector cell population present within peripheral blood leukocytes (PBLs) was composed of agranular lymphocytes that formed conjugates with, and induced apoptosis in, allogeneic target cells. Both JAM and TUNEL assays showed that PBLs induced target cell DNA fragmentation within 1 h of coculture. In addition, fixed effectors did not induce target cell necrosis or apoptosis, and target cell lysis was completely inhibited by chelation of free Ca(2+) by EGTA. These results suggest that catfish peripheral blood-derived effector cells utilize a secretory mechanism rather than a ligand-based mechanism to trigger apoptosis.     

Cytotoxic T lymphocyte sequential killing of immobilized allogeneic tumor target cells measured by time-lapse microcinematography.

Sequential killing of allogeneic target cells by immune cytotoxic T lymphocytes (CTL) was directly observed by time-lapse microcinematography. Target cells (EL4 lymphoma cells from C56BL/6 mice), coated with Fab fragments of goat antibody to EL4, were immobilized by binding to the floor of a polystyrene tissue culture flask that had been precoated with specifically purified anti-goat Fab. On adding immune BALB/c spleen CTL to such target cell monolayers it could be verified by direct observation that individual CTL could sequentially kill several target cells, that the CTL usually separated from the target cell before target cell death, that not all contacted target cells were killed, and that duration of contact was variable and not correlated with subsequent target cell death.     

Pulsed electric field inactivation of diarrhoeagenic Bacillus cereus through irreversible electroporation

The physical effects of high-intensity pulsed electric fields (PEF) on the inactivation of diarrhoeagenic Bacillus cereus cells suspended in 0.1% peptone water were examined by transmission electron microscopy (TEM). The levels of PEF-induced microbial cell death were determined by enumeration on tryptone soy yeast extract agar and Bacillus cereus-selective agar plates. Following exposure to lethal levels of PEF, TEM investigation revealed irreversible cell membrane rupture at a number of locations, with the apparent leakage of intracellular contents. This study provides a clearer understanding of the mechanism of PEF-induced cellular damage, information that is essential for the further optimization of this emerging food-processing technology.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. I. Resolution and characterization.

Addition of high molecular weight dextran to culture medium prevents the initiation of T lymphocyte-mediated killing by holding the cytolytic T lymphocytes (CTL) and target cells in suspension and preventing intercellular contact. Suspension in 10% dextran was used to interrupt the ongoing formation of adhesions between CTL and target cells already in contact in a centrifuged pellet. The results demonstrate that 1) firm adhesions form between CTL and target cells within 1 min at 37 degrees C; 2) once formed, these adhesions are stable at low temperature and are resistant to mechanical shearing forces; 3) these adhesions can be disrupted by EDTA; 4) immediately after the adhesions form, separation of the CTL from the target cells prevents lysis of the latter; 5) after incubation of targets adhering to CTL for an additional 6 min at 37 degrees C, removal of the CTL no longer prevents target cell lysis. Thus, target cells become "programmed" for subsequent lysis within a few minutes after contact with CTL, after which lysis occurs during the next several hours without further participation of the effector cell. At 15 degrees C, adhesions form 1/17 as fast as at 37 degrees C. Programming of target cells for lysis occurs 1/76 as fast at 15 degrees C as at 37 degrees C. Thus, the programming for lysis step is about 4-fold more temperature dependent than the adhesion step. In addition to being detected by subsequent target cell lysis in 10% dextran, the adhering cell clusters can be counted with low power microscopy. This permitted verification that EDTA separates the clusters after programming for lysis is complete. Moreover, the great majority of the clusters seen at 37 degrees C are antigen-specific. Knowledge of the cluster size distribution and the subsequent level of lysis permits the deduction that not less than 6% of the sensitized peritoneal cell populations used were CTL.     

Unexpected cell surface labeling in conjugates between cytotoxic T lymphocytes and target cells

Specific binding of target cells by cytotoxic T lymphocytes (CTL) is an example of tight interaction between two different cell types. The molecular events that occur at the cell membranes during these interactions are largely unknown. In the present report, we describe an electron microscopic immunostaining study made on CTL-target cell conjugates. Various membrane structures were labeled with monoclonal antibodies specific for structures possibly relevant to cytolysis (Lyt-2, LFA-1, and target cell class I major histocompatibility antigens) or probably unrelated to the cytolytic process (effector cell class I major histocompatibility antigens). Antibodies against Thy-1 were also used. Staining was achieved with immunoperoxidase or immunoferritin. With both techniques nonconjugated cells were either stained or not, depending on whether they bore the antigen corresponding to the antibody used. However, when conjugated to an antigen-bearing cell, a "non-antigen bearing" cell was labeled near the cell interaction area. No increased Fc receptor activity could be detected on bound cells near the interaction area. These data are consistent with the occurrence of limited exchange of membrane macromolecules between bound CTL and target cell.     

Isolation and characterization of gap junctions from tissue culture cells.

The purification of membrane proteins in a form and amount suitable for structural or biochemical studies still remains a great challenge. Gap junctions have long been studied using electron microscopy and X-ray diffraction. However, only a limited number of proteins in the connexin family have been amenable to protein or membrane purification techniques. Molecular biology techniques for expressing large gap junctions in tissue culture cells combined with improvements in electron crystallography have shown great promise for determining the channel structure to better than 10 A resolution. Here, we have isolated two-dimensional (2D) gap junction crystals from HeLa Cx26 transfectants. This isoform has never been isolated in large fractions from tissues. We characterize these preparations by SDS-PAGE, Western blotting, negative stain electron microscopy and atomic force microscopy. In our preparations, the Cx26 is easily detected in the Western blots and we have increased expression levels so that connexin bands are visible on SDS-PAGE gels. Preliminary assessment of the samples by electron cryo-microscopy shows that these 2D crystals diffract to at least 22 A. Atomic force microscopy of these Cx26 gap junctions show exquisite surface modulation at the extracellular surface in force dissected gap junctions. We also applied our protocol to cell lines such as NRK cells that express endogenous Cx43 and NRK and HeLa cell lines transfected with exogenous connexins. While the gap junction membrane channels are recognizable in negatively stained electron micrographs, these lattices are disordered and the gap junction plaques are smaller. SDS-PAGE and Western blotting revealed expression of connexins, but at a lower level than with our HeLa Cx26 transfectants. Therefore, the purity and morphology of the gap junction plaques depends the size and abundance of the gap junctions in the cell line itself.     

2-Methoxyestradiol-bis-sulfamate induces apoptosis and autophagy in a tumorigenic breast epithelial cell line

In anticancer research where the focus is on finding agents that induces cell death while leaving non-tumorigenic cells less affected, a novel 2-methoxyestradiol derivative has come forth. 2-Methoxyestradiol-bis-sulfamate (2-MeOE2bisMATE) is a 2-methoxyestradiol derivative produced by bis-sulphamoylation, which possesses increased antiproliferative activity and biological availability. Several questions remain regarding the type of cell death mechanisms and possible induction of autophagy by 2-MeOE2bisMATE. The aim of this in vitro study was to investigate the cell death mechanisms exerted by 2-MeOE2bisMATE in an adenocarcinoma cell line (MCF-7) by analyzing its influence on cell growth, morphology, and possible induction of cell death. Spectrophotometry (crystal violet staining), transmission electron microscopy (TEM), light microscopy (hematoxylin and eosin staining), and fluorescent microscopy (Hoechst 33342, propidium iodide and acridine orange) were employed. Spectrophotometrical studies indicated that 2-MeOE2bisMATE decreased cell numbers to 75% in MCF-7 cells after 24 h and to 47% after 48 h of exposure. TEM demonstrated membrane blebbing, nuclear fragmentation, and chromatin condensation indicating the hallmarks of apoptosis. Light microscopy revealed the presence of several cells blocked in metaphase, and apoptotic cells were also observed. Fluorescent microscopy demonstrated increased lysosomal staining; suggesting the induction of autophagy. 2-MeOE2bisMATE shows therapeutic potential, as an, anticancer agent, and the investigation of the cell death mechanisms used by 2-MeOE2bisMATE, thus, warrants further investigation.     

Structural characterization of a rat acinar cell tumor

A transplantable acinar cell tumor of the rat pancreas has been examined by light and electron microscopy. The tumor cells, though highly cytodifferentiated and characterized by the presence of abundant rough-surfaced endoplasmic reticulum, elements of the Golgi complex, and zymogen granules, undergo mitosis in a manner similar to that seen in the developing pancreas. Cells in the parenchyma of the tumor grow as disarrayed cords and sheets, are randomly oriented with respect to each other, and do not form acinar structures. However, when in contact with the adventitial surface of blood vessels, the tumor cells palisade and form a polarized layer of cells with their zymogen granule-rich poles oriented away from the vessel lumen. Only in this area of the tumor is a basal lamina present that underlies the basal plasmalemma of the reoriented epithelial cells. Freeze-fracture electron microscopy of tumor cells in the parenchyma shows extensive disruption of tight junctions whose sealing strands are randomly distributed over the entire plasmalemma. Gap junctions are infrequent and when present are often enclosed by tight-junctional strands. Intramembrane particles are randomly distributed over the cell surface. Both the absence of basal lamina and derangement of the junctional complexes may account in part for the altered morphogenesis of this tumor.     

Intercellular connectivity in the eight-cell Xenopus embryomcorrelation of electrical and morphological investigations.

The distribution of individual intercellular electrical junctions has been examined in eight-cell Xenopus embryos using linear systems analysis. Morphological evidence for corresponding intercellular contacts has been sought by light microscopy and scanning electron microscopy. The electrical investigation indicated that each cell is directly coupled to each of the other seven cells by identical resistive junctions. Scanning electron microscopy of the cell surfaces of cleaved embryos revealed protrusions from the surfaces of the cells which could mediate such intercellular connections. Light microscopy of serial sections through the embryos also showed fine processes of the cell surfaces which come into contact with several other cells. The complete intercellular connectivity suggested by these results appears to be an extension of similarly close connectivity in the two- and four-cell embryos. The possible significance of this high connectivity to morphogenesis is discussed.     

Mechanism of killing by virus-induced cytotoxic T lymphocytes elicited in vivo.

The mechanism of lysis by in vivo-induced cytotoxic T lymphocytes (CTL) was examined with virus-specific CTL from mice infected with lymphocytic choriomeningitis virus (LCMV). LCMV-induced T cells were shown to have greater than 10 times the serine esterase activity of T cells from normal mice, and high levels of serine esterase were located in the LCMV-induced CD8+ cell population. Serine esterase was also induced in purified T-cell preparations isolated from mice infected with other viruses (mouse hepatitis, Pichinde, and vaccinia). In contrast, the interferon inducer poly(I.C) only marginally enhanced serine esterase in T cells. Serine esterase activity was released from the LCMV-induced T cells upon incubation with syngeneic but not allogeneic LCMV-infected target cells. Both cytotoxicity and the release of serine esterase were calcium dependent. Serine esterase released from disrupted LCMV-induced T cells was in the form of the fast-sedimenting particles, suggesting its inclusion in granules. Competitive substrates for serine esterase blocked killing by LCMV-specific CTL, but serine esterase-containing granules isolated from LCMV-induced CTL, in contrast to granules isolated from a rat natural killer cell tumor line, did not display detectable hemolytic activity. Fragmentation of target cell DNA was observed during the lytic process mediated by LCMV-specific CTL, and the release of the DNA label [125I]iododeoxyuridine from target cells and the accompanying fragmentation of DNA also were calcium dependent. These data support the hypothesis that the mechanism of killing by in vivo-induced T cells involves a calcium-dependent secretion of serine esterase-containing granules and a target cell death by a process involving nuclear degradation and DNA fragmentation.     

Reorientation and fusion of cytotoxic T lymphocyte granules after interaction with target cells as determined by high resolution cinemicrography

The interaction of murine cytotoxic T lymphocyte (CTL) clones with human lymphoblastoid target cells was studied in thin preparations by using high resolution cinemicrography. CTL not bound to target cells were morphologically polar, possessing a broad leading edge containing the nucleus, and a tapered tail containing a large number of granules. The CTL were observed to move by the extension of pseudopods from the leading edge. Initial contact with a target cell was made via the leading edge of the CTL. If the human target cell expressed the appropriate HLA antigen, distinct morphologic changes occurred in the CTL as early as 2 min after initial contact. The CTL rounded up, and the nucleus moved from a position adjacent to the zone of contact to be replaced by the cytoplasmic granules. Redistribution of the granules was completed as early as 10 min after initial contact was made. These morphologic changes did not occur when the CTL made contact with other CTL, or with target cells that did not express the appropriate HLA antigens. In studies that make use of Nomarski optics, an apparent fusion of CTL cytoplasmic granules with the membrane in the vicinity of the target cell contact area was observed 4 min after binding, and before granule reorientation was complete. These data provide direct evidence for the occurrence of both reorientation of the cytoplasmic contents and granule fusion in CTL with a time course similar to that of administration of the lethal hit.     

Endocytosis of a cytotoxic human high density lipoprotein results in disruption of acidic intracellular vesicles and subsequent killing of African trypanosomes

The host range of Trypanosoma brucei brucei is restricted by the cytolytic effects of human serum high-density lipoprotein (HDL). The lytic activity is caused by a minor subclass of human serum HDL called trypanosome lytic factor (TLF). TLF binds in the flagellar pocket to specific TLF-binding sites. Internalization and localization of TLF to a population of endocytic vesicles, and ultimately large lysosome-like vesicles, precedes lysis of T. b. brucei. The membranes of these large vesicles are disrupted by the accumulation of TLF particles. Inhibitor studies with lysosomotropic amines have shown these large vesicles to be acidic in nature and that prevention of their rupture spares the cells from TLF-mediated lysis. Furthermore, leupeptin inhibition suggests that a thioprotease may be involved in the mechanism of TLF- mediated lysis of T. b. brucei. Based on these results, we propose a lytic mechanism involving cell surface binding, endocytosis and lysosomal targeting. This is followed by lysosomal disruption and subsequent autodigestion of the cell.     

Human monocyte-mediated lysis of antibody-coated tumor cells: the role of the cytoskeleton in monocytes

Human monocytes (M phi) show high cytolytic activity towards antibody-coated tumor cells (AbK562). In this report, the relationship between the cytoskeleton in the M phi and the M phi cytolytic activity has been investigated. The actin filament inhibitors cytochalasin B and dihydrocytochalasin B (H2CB) both reduced M phi-mediated lysis of AbK562 cells by approximately 50% at a concentration of 1 microM. This concentration of H2CB did not inhibit the number of target cells bound to M phi. Dihydrocytochalasin B did not inhibit the M phi ability to release cytotoxic protein factors, suggesting that H2CB does not inhibit lysis by inhibiting release of cytotoxic protein factors. Immunofluorescence microscopy showed a rapid accumulation of actin filaments towards the contact area in more than 80% of the examined M phi-AbK562 conjugates. Exposure to H2CB did not prevent this accumulation, but caused aggregation of the accumulated actin filaments in the contact area with the target cell. Accumulation of actin filaments did not occur toward tumor cells not coated with antibodies. Scanning and thin section electron microscopy demonstrated large M phi pseudopodia directed toward the AbK562 cells, with close apposition of the effector and target cell membranes with interdigitations. The formation of the M phi pseudopodia was inhibited by exposure to H2CB. These observations indicate that M phi membrane motility toward AbK562 cells is closely related to M phi-mediated lysis of AbK562 cells. Immunofluorescence microscopy of the microtubule-organizing center (MTOC) and the Golgi apparatus revealed that both the MTOC and the Golgi apparatus in M phi reoriented towards the bound AbK562 cells in approximately 45% of the examined M phi-AbK562 conjugates. The microtubule-depolymerizing drugs colchicine and vinblastine did not inhibit M phi-mediated lysis of AbK562 cells at concentrations which disrupted the microtubule arrays in the M phi. The carboxylic ionophore monensin, which blocks Golgi-derived secretion, inhibited M phi-mediated lysis of AbK562 to a lesser extent as compared to H2CB. These results suggest that microtubule functions are of less importance in M phi-mediated lysis of AbK562 cells as compared to actin filament functions. However, the MTOC and the Golgi apparatus could participate in M phi-mediated lysis of AbK562 cells by mechanisms related to secretion of cytotoxic molecules.     

Studies on the induction and expression of T cell-mediated immunity. XIV. Antigen-nonspecific oxidation-dependent cellular cytotoxicity (ODCC) mediated by sodium periodate oxidation of cytotoxic T lymphocytes

Alloimmune murine thymus-derived cytotoxic lymphocytes (CTL) generated in vivo or in vitro are shown to lyse antigen-nonspecific target cells (tumor cells, Con A, and LPS blasts) following treatment of CTL with an oxidizing agent, sodium periodate (NaIO4). It has been shown that NaIO4 oxidizes terminal sialic acid residues of cell surface macromolecules. The presence of reactive aldehyde groups, generated by NaIO4 modification, is required for the expression of antigen-nonspecific cytotoxicity because treatment of modified cells with a reducing agent such as potassium borohydride (KBH4) resulted in the abrogation of cytotoxicity. However, KBH4 treatment of unmodified or NaIO4-modified CTL has no effect on antigen-specific cytotoxicity. The modification of CTL by NaIO4 is sufficient to lead to the formation of lymphocyte-target cell conjugates and lysis of bound targets. Monoclonal antibodies directed against the Lyt-2 antigens of CTL, but not Lyt-1 antigens, in the absence of complement inhibited the nonspecific cytotoxicity resulting from NaIO4 modification of effector lymphocytes. These findings suggest that the mere interaction with or perturbation of appropriate cell surface molecule(s) of effector lymphocytes such as Lyt antigens by receptor-ligand interaction in SCMC or by NaIO4 modification in ODCC may lead to the expression of cytotoxicity. The present studies demonstrate a functional role of surface carbohydrates on CTL in cell-to-cell recognition and interactions. Furthermore, the results suggest that target cell modification is not a requisite for recognition and lysis in an antigen-nonspecific cytotoxic system such as ODCC. However, partial blocking of ODCC by alloantibodies directed against the H-2 of unmodified target cells suggests that NaIO4-modified CTL recognize unrelated target H-2 antigens. The implication of these findings on the molecular mechanism of cell-mediated cytotoxicity is discussed.