The incorporation of glucose into alpha-1,4-glucan proteins of bovine retina membranes.

—Incubation of bovine retina membranes with UDP-[¹⁴C]glucose resulted in the incorporation of [¹⁴C]glucose into endogenous α-1, 4-glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m -sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein-I (GP-I), insoluble in TCA, and glucan protein-II (GP-II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP-I and GP-II were precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA). A third fraction, glucan protein-III (GP-III) was found when incubations were carried out with UDP-[¹⁴C]glucose at millimolar instead of micromolar concentrations. GP-III was soluble in TCA and in TCA-PTA and precipitable by ethanol from the TCA soluble fraction. GP-II was excluded from a Sephadex G-200 column and showed a greater size than GP-I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G-25 column and were of a greater size than the labelled residues released with salivary α-amylase. Only radioactive maltose was found after a-amylase treatment. When membranes containing labelled GP-I and GP-II were incubated with unlabelled UDP-glucose at millimolar concentrations, GP-I was converted into GP-II and GP-III was formed.     

Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.     

Fractionation and Characterization of the Sulfated Oligosaccharide Chains of Ovomucin

The O-glycosidically linked carbohydrate units of ovomucin were released from serine and threonine in peptide as oligosaccharide chains by alkali treatment with and without borohydride. Two sulfated oligosaccharides were fractionated by using gel filtration and ion-exchange chromatography. The yield of sulfated oligosaccharides released by alkali treatment was higher in the presence of borohydride than in the absence of borohydride. The sulfated oligosaccharides released by alkali treatment with borohydride were as follows: an oligosaccharide composed of N-acetylgalactosaminitol, galactose, N-acetylneuraminic acid and sulfate in a molar ratio of about 1: 1: 1: 1 and another oligosaccharide in a molar ratio of about 1:1: 0.6: 0.5.     

A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins.

O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.     

Phenotypic and Functional Characteristics of Blood Natural Killer Cells from Melanoma Patients at Different Clinical Stages

Melanomas are aggressive skin tumors characterized by high metastatic potential. Immunotherapy is a valuable alternative for metastatic melanoma patients resistant to chemotherapy. Natural Killer (NK) cells are efficient anti-tumor cytotoxic effectors. We previously showed that blood NK cells from stage IV metastatic melanoma patients display decreased NK receptors and that chemotherapy modifies the functional status of blood NK cells. To investigate the role of NK cells along melanoma progression, we have here studied NK cells from patients at different stages of the disease. First, we showed that ex vivo NK cells from certain stage III–IV patients displayed low degranulation potential. Using a dynamic label-free assay, we found that immunoselected IL-2 activated blood NK cells from patients efficiently lysed melanoma cells through NKp46 and NKG2D receptors, independently to the clinical stage. Moreover, the ex vivo phenotype of circulating NK cells from 33 patients (stage I to IV) was extensively analyzed. NK cells from patients displayed higher variability in the percentages of Natural Cytotoxicity Receptors (NCR) and Natural Killer Group 2D (NKG2D) receptor expression compared to donor NK cells. The main defect was the decreased expression of NCR1 (NKp46) by NK cells from metastatic patients. Interestingly, we found a positive correlation between the NK cell percentages of NKp46 and the duration of stage IV in melanoma patients. Finally, we showed that NK cells infiltrated primary melanomas and displayed a predominant peritumoral distribution. These results are new arguments for the development of NK-based therapies in melanoma patients.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.     

Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.     

Structure determination of five sulfated oligosaccharides derived from tracheobronchial mucus glycoproteins

The structure of five sulfated oligosaccharide units of highly anionic tracheobronchial mucous glycoproteins, isolated from a cystic fibrosis patient's sputum, were established. Reduced oligosaccharides (84%) were released under alkaline borohydride conditions, and the acidic oligosaccharides (63%) were isolated by Dowex 1-X2 chromatography. Following the removal of acidic oligosaccharides possessing N-acetylneuraminic acid and L-fucose by lectin affinity chromatography a heterogeneous mixture of sulfated oligosaccharides was obtained. From this fraction, five short chain monosulfated oligosaccharides (S-I to S-V) were purified by sequential separation by SynChroprep AX300 anion exchange high pressure liquid chromatography, gel filtration on Bio-Gel P-2, and high voltage paper electrophoresis. Based on the results of carbohydrate composition, sequential exoglycosidase degradation, permethylation analysis, lectin affinity chromatography, and periodate oxidation, the following structures (where GalNAcol is N-acetylgalactosaminitol) were proposed for these oligosaccharides. (formula; see text)     

Carbohydrate structures of HVJ (Sendai virus) glycoproteins

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fucose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharides by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gal beta 1 leads to (Fuc alpha 1 leads to 3) GlcNAc group in their outer chain moieties.     

Expansion of NK Cells and Reduction of NKG2D Expression in Chronic Lymphocytic Leukemia. Correlation with Progressive Disease

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL.     

Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56(+)granulysin(+) cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.     

Effect of azacytidine in the release of leukemia inhibitory factor,oncostatin m,interleukin (IL)-6, and IL-11 by mononuclear cells of patients with refractory anemia

5-azacytidine (AZA) yields hematologic improvement in patients with myelodysplastic syndromes (MDS). Ineffective hemopoiesis in MDS produce the paradox of high intramedullary cellularity with peripheral cytopenias. Leukemia inhibitory factor (LIF), oncostatin M (OSM), interleukin (IL)-6, and IL-11 regulate hemopoiesis and LIF, OSM, and IL-6 also inhibit the proliferation of myeloid leukemic cell lines through the signal-transducing subunit gp130. These IL-6-type cytokines were measured by enzyme-linked immunosorbent assay in cell culture supernatants (SN) obtained from peripheral blood mononuclear cells (MNC) and monocyte-depleted MNC of patients with refractory anemia (RA; n=12) and healthy individuals (n=10). AZA down-regulated OSM, IL-6, and IL-11 release by MNC of patients but not by MNC from healthy individuals. Patient's SN had significantly lower concentrations of LIF, OSM, and IL-11 than SN of normal subjects. When monocyte-depleted MNC of patients were stimulated with phytohemagglutinin a significant increment in OSM levels was observed. In contrast, monocyte depletion in healthy subjects did not cause any significant change in OSM values. We conclude that: (a) AZA inhibits the release of OSM, IL-6, and IL-11 exclusively in RA-diseased MNC, (b) Patient's MNC release subnormal amounts of LIF, OSM, and IL-11, and (c) RA-derived monocytes probably down-regulate OSM release by phytohemagglutinin-activated MNC.     

Elucidation of an essential structure recognized by an anti-GalNAc alpha-Ser(Thr) monoclonal antibody (MLS 128).

To determine the epitopic structure for an anti-GalNAc alpha-Ser(Thr) (anti-Tn) monoclonal antibody, MLS 128, asialo-ovine submaxillary mucin was digested with various proteases, and the digests were fractionated by immunoaffinity column chromatography and high performance liquid chromatography. From the tryptic digest, a glycopeptide, GP-I, and five other glycopeptides, GP-1-5, were obtained as bound and unbound fractions, respectively, of the immunoaffinity column. By solid phase radioimmunoassaying, it was found that GP-I was strongly immunoreactive, whereas GP-1-5 were poorly immunoreactive. On treatment with V8 protease, GP-I was converted to two glycopeptides, one with poor reactivity and the other with intermediate reactivity. From the thermolysin digest, the smallest fragment, GP-II, was isolated, which was as strongly immunoreactive as GP-I. GP-II corresponded to a part of GP-I, its sequence being Leu-Ser*-Glu-Ser*-Thr*-Thr*-Gln-Leu-Pro-Gly, where asterisks denote amino acids to which an alpha-GalNAc residue is attached. Other anti-Tn monoclonal antibodies, NCC-LU-35 and CA 3239, showed essentially the same reactivity to these glycopeptides as MLS 128 did. The glycopeptides (GP-1-5), which exhibited poor immunoreactivity, contained various GalNAc-containing structures, such as GalNAc-Ser, GalNAc-Thr, GalNAc-Ser-(GalNAc)-Ser, and GalNAc-Thr-(GalNAc)-Thr. These results indicate that a glycopeptide including a cluster structure, Ser*-Thr*-Thr*, is an essential part of the epitope recognized by anti-Tn antibodies.     

Comparison of the cytotoxic response against ovarian cancer by immune effector cells isolated and expanded from normal donors and ovarian cancer patients

Background/AimsThe aim of this study was to compare the cytotoxic response against ovarian cancer (OC) cells elicited by different immune effector cells in combination with the cytokines interleukin (IL)-2 and interferon (IFN) α-2b.MethodsOC cells were co-cultured with peripheral blood mononuclear cells (PBMC) from normal donors or OC patients and IL-2 or IFN α-2b alone or in combination, in order to determine the cytotoxicity. T cells were isolated from healthy donors to determine T cell cytotoxic activity. PBMC from healthy donors and OC patients were expanded in an IL-2/IL-7/IL-12 cocktail with and without anti-CD3 antibody, and the cytotoxic activity measured. Flow cytometry was performed on primary, selected and expanded cells to determine T, B, and natural killer- (NK) cell percentages.ResultsHealthy donor PBMC elicited a significant cytotoxic response (59%) compared with OC patient PBMC (7%). T cells enriched from normal donors elicited a significant cytotoxic response (18%) compared with controls lacking effector cells (1.4%); however, the cytotoxicity observed was significantly less compared with unselected PBMC. Expanded effector cells consisted primarily of T cells (98%) and the fold-expansion was significantly higher in the presence of anti-CD3 (19- versus 132-fold). No significant difference in the expansion (either fold-expansion or cell type) was observed between OC patients and healthy donors. Expanded cells from both healthy donors and OC patients elicited a significant cytotoxic response in the presence of IL-2 (19% and 22%) compared with controls.ConclusionsPBMC from OC patients do not elicit a significant cytotoxic response; however, ex vivo-expanded cells from OC patients are capable of cytotoxic killing similar to unexpanded T cells isolated from normal donors. These data provide the groundwork for further development of cellular therapy against OC.     

The structure of carbohydrate chains of blood-group substance. Isolation and elucidation of the structure of higher oligosaccharides from blood-group substance H

Twenty individual higher reduced oligosaccharides, having from seven to eleven monosaccharide units, were isolated after sodium borohydride degradation of blood-group substance H from pig stomach linings. Anion-exchange high-pressure liquid chromatography appears to be a very convenient and effective method for this kind of higher oligosaccharide mixtures separation. The oligosaccharide structures were determined by means of periodate oxidation, methylation analysis, partial acid and enzymic hydrolysis. It has been found that all the oligosaccharides investigated can be divided into four series. The oligosaccharides belonging to each series have the common oligosaccharide fragment to which terminal L-fucose and/or N-acetyl-D-glucosamine residues are attached. Comparison of all the oligosaccharide structures, including tri, penta and hexasaccharides described earlier, shows that the lower oligosaccharides represent the structural element of the higher oligosaccharides.     

Glycosylation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acid analogues and by tunicamycin

Addition of asparagine-linked oligosaccharides to nascent murine leukemia virus (MuLV)-encoded membrane glycoproteins was inhibited either completely by tunicamycin or specifically at Asn-X-Thr glycosylation sites by incorporation of the threonine analogue beta-hydroxynorvaline. In conditions of partial analogue substitution, a series of subglycosylated components is formed which are related by a constant apparent Mr difference when assayed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The total number of asparagine-linked oligosaccharides is then estimated by dividing the measured apparent Mr of one oligosaccharide into the total apparent Mr difference between the complete glycoprotein and the polypeptide chain that is synthesized in cells incubated with tunicamycin. Correct results were obtained using glycoproteins with known numbers of oligosaccharides. Our analyses indicate that the gp70 membrane envelope glycoproteins of certain ecotropic MuLVs contain seven oligosaccharides, whereas the GIX+ antigen-containing variant gp70 contains one fewer Asn-X-Thr-linked oligosaccharide. The membrane glycoprotein encoded by the gag gene of Friend MuLV contains only one asparagine-linked oligosaccharide. Similarly, the gp55 membrane glycoprotein encoded by Friend erythroleukemia virus contains four asparagine-linked oligosaccharides. Pulse-chase and cell surface iodination analyses indicate that MuLV membrane envelope glycoprotein processing by partial proteolysis and transport to the cell surface can be efficiently blocked by structural perturbations caused by incorporation of different amino acid analogues or by loss of oligosaccharides. Our data also suggest that loss of oligosaccharides may expose new antigenic sites in viral membrane glycoproteins and increase their susceptibility to intracellular proteolysis.     

The combination of normal-phase and reverse-phase high-pressure liquid chromatography with NMR for the isolation and characterization of oligosaccharide alditols from ovarian cyst mucins

Normal-phase high-pressure liquid chromatography (HPLC) on amino-bonded silica with elution by aqueous acetonitrile is shown to be an especially suitable complement to reverse-phase HPLC on octadecyl silica for the fractionation of oligosaccharide alditols produced by alkaline borohydride degradation of mucin glycoproteins. The former technique separates well on the basis of molecular size, while the latter method shows selectivity for stereoisomers. Stereoisomeric pairs of tetra-, penta- and hexasaccharide alditols show relative retention times ranging from 3 to 12, resulting in excellent preparative separations in reverse-phase chromatography. From a single ovarian cyst glycoprotein, H and Lewis b active, 13 oligosaccharides, representing essentially the entire carbohydrate content, have been isolated. The structures of 12 of the oligosaccharides have been determined by ¹H-NMR spectroscopy. For those oligosaccharides which have been isolated from other sources and whose NMR spectra have been previously reported, unambiguous structural identification follows directly. Structures of oligosaccharides differing by only one or two residues from those whose NMR spectra are known may be deduced by a simple algorithm utilizing chemical shift analogies.     

Surface receptors and immune activity of purified human circulating mononuclear cells. III. The mechanisms of lysis, by lymphocytes and monocytes, of target cells in the lymphocyte-dependent and monocyte-dependent ADCC, NK, NOCC, and MICC cytotoxic reactions

The abilities of unfractionated mononuclear cells (MNC), monocytes (98-99% pure), and lymphocytes (98-99% pure) to carry out the lysis of target cells in the ADCC, NK, NOCC, and MICC assays were compared. Lymphocytes by themselves were able to lyse the CRBC (ADCC), K-562 (NK), and RRBC (MICC) target cells. The monocytes were very effective in the lysis of the CRBC (MICC) target cells. However, the lysis of two other target cells--RRBC (NOCC) and HRBC (ADCC)--required the simultaneous presence of both lymphocytes and monocytes in order to effect optimal lysis. Soluble factor(s) secreted by the cytotoxic cells capable of lysing the target cells were detected only in the NK assay. The activity of the soluble cytotoxic factor (NKCF) was only 25-40% of that exhibited by the cytotoxic NK cells and it was secreted by the cytotoxic cells after 48 hr of culture and not 24 hr of culture which is the usual assay condition. The NKCF was cytotoxic only to the NK target cells and not to the target cells used in the ADCC, NOCC, and MICC cytotoxic assays. Different classes of lymphocytes were cytotoxic in the monocyte-independent assays [ADCC (CRBC), NK (K-562), and MICC (RRBC)]. The null lymphocytes and the T lymphocytes were the primary cytotoxic cells in the ADCC and MICC assays, respectively, whereas the T, B, and null cells were almost equally cytotoxic in the NK assay. With respect to the monocyte-dependent assays [ADCC (HRBC), NOCC (RRBC), and MICC (CRBC)], the cytotoxic activity of any one class of lymphocytes failed to approach that of the unfractionated MNC. The T cells were the most cytotoxic; the B cells exhibited limited cytotoxic activity in only the ADCC assay and the null cells showed no cytotoxic activity. However, the combination of T and non-T cells and, to a lesser extent, T and B cells, exhibited much greater cytotoxic activity than the individual cells and together were as cytotoxic as the unfractionated MNC. It is concluded that, depending upon the selection of the target cells, lysis in the ADCC, NK, NOCC, and MICC assays may be effected by lymphocytes only, by monocytes only, by both monocytes and lymphocytes, or as a result of lymphocyte-monocyte collaboration. In the latter instance more than one class of lymphocytes must be present in order for maximum cytotoxic activity to be expressed.