NK Cells and Other Natural Effector Cells

otherwise.

I

CHARACTERISTICS OF NK CELLS

A

LARGE GRANULAR LYMPHOCYTES

1

HUMAN

MORPHOLOGY AND CYTOCHEMISTRY OF HUMAN LARGE GRANULAR LYMPHOCYTES¹

Carlo Enrico Grossi,     Department of Human Anatomy, University of Genoa, Genoa, Italy

Manlio Ferrarini,     Department of Clinical Immunology, University of Genoa, Genoa, Italy

Publisher Summary

This chapter describes morphology and cytochemistry of human large granular lymphocytes (LGLs) or natural killer (NK) cells. LGL represent the bulk of two mononuclear cell subsets of the human peripheral blood, namely, the TG cells (cells forming rosettes with sheep erythrocytes and expressing avid receptors for the Fc portion of IgG) and the third populations cells (TPC). LGL appear as medium-sized lymphocytes with round or indented nuclei, clumped chromatin, and usually prominent nucleoli. The cytoplasm is abundant besides rare mitochondria and isolated profiles of the rough endoplasmic reticulum (RER). LGL contain several acid hydrolases, which are a specific marker of the lysosomal system. They are alpha-naphthyl acetate (ANAE) and -butirate (ANBE) esterase, acid phosphatase (AP), and beta-glucuronidase (B-Gluc). With azo-dye technique at the light microscope, these enzymes give a positive reaction in the paranuclear region, with a granular or more often diffuse pattern of staining. With a Gomori-type of substrate for AP, only a granular reaction is obtained with the positivity mainly concentrated in the paranuclear area. These findings suggest that the acid hydrolases could be in the granules and, possibly, in the coated vesicles, which are more numerous in the paranuclear area.

INTRODUCTION

Human peripheral blood cells with a lymphoid morphology and intracytoplasmic azurophilic granules have been described long ago and termed monocytoid or leukocytoid lymphocytes (Pappenheim and Ferrata, 1911). However, only recently have they been recognized as a distinct subset of mononuclear cells (Grossi et al., 1978; Ferrarini et al., 1980) with specialized functions, such as antibody-dependent cell cytotoxicity (ADCC) and natural killer (NK) activity (Timonen et al., 1981). These cells are usually referred to as Large Granular Lymphocytes (LGL) or NK cells.

LGL represent the bulk of two mononuclear cell subsets of the human peripheral blood, namely, the TG cells (i.e., cells forming rosettes with sheep erythrocytes and expressing avid receptors for the Fc portion of IgG) and the third populations cells (TPC) (Ferrarini et al., 1980). TPC are defined as those null cells (i.e., cells not forming rosettes with sheep erythrocytes and not expressing surface immunoglobulin) with avid Fc receptors (Winchester et al., 1975).

Here we shall describe the morphological and cytochemical features of LGL, with a special emphasis on the granules and on the mechanism of their formation. These studies revealed possible stages of LGL maturation and this approach seems further exploitable for the search of LGL precursors.

ULTRASTRUCTURE OF PERIPHERAL BLOOD LGL

Highly enriched LGL preparations have been used for morphological studies. Such enrichment has been obtained with three different procedures: 1) isolation of TG cells (Grossi et al., 1978); 2) separation of TPC (Ferrarini et al., 1980); and 3) purification of LGL on Percoll gradients (Timonen et al., 1981). No differences in the results were obtained with these various preparations.

LGL appear as medium-sized lymphocytes with round or indented nuclei, clumped chromatin and usually prominent nucleoli. The cytoplasm is abundant and, besides rare mitochondria and isolated profiles of the rough endoplasmic reticulum (RER), displays the following distinctive features (Grossi et al., 1982): a) an extended Golgi apparatus which buds smooth vesicles surrounding stacks of parallel Golgi tubules and saccules; b) numerous coated vesicles which are sparse in the cytoplasm or, more often, concentrated in the nuclear notch; c) unit membrane-bound granules containing a matric of variable density; these organelles are likely to correspond to the azurophilic granules of the Giemsa-stained cells; d) the remarkable absence of phagolysosomes (Fig. 1).

FIGURE 1 Ultrastructure characteristics of human peripheral blood LGL. a) Section of a whole LGL illustrating the overall features of this cell type. The inset shows a Giemsa-stained LGL containing numerous azurophilic granules. b) The Golgi region of a LGL. The arrow points to a group of coated vesicles. c) Ultrastructural characteristics of the mature LGL granules. d) Ultrastructure of two PTA. The organelle seen on the left is bounded by a unit membrane whereas the array of transversely-sectioned tubules on the rights is free in the cytoplasm.

Recently it has been suggested that parallel tubular arrays (PTA) which may be common in LGL (Costello et al., 1980; Payne and Glasser, 1981) could correspond to the azurophilic granules. However, in our experience, PTA are observed in a minority of cells only and therefore we believe that (i) PTA cannot be used as a marker for LGL identification and (ii) PTA do not correspond to the azurophilic granules (Fig. 1).

THE LYSOSOMAL SYSTEM OF PERIPHERAL BLOOD LGL

LGL contain several acid hydrolases, which are a specific marker of the lysosomal system. They are alpha-naphthyl acetate (ANAE) and -butirate (ANBE) esterase, acid phosphatase (AP) and beta-glucuronidase (B-Gluc) (Grossi et al., 1978, Saksela et al., 1979; Ferrarini et al., 1980; Costello et al., 1980). With azo-dye technique at the light microscope, these enzymes give a positive reaction in the paranuclear region, with a granular or more often diffuse pattern of staining. With a Gomori-type of substrate for AP, only a granular reaction is obtained with the positivity manily concetrated in the paranuclear area (Grossi et al., 1982). These findings suggest that the acid hydrolases could be in the granules and, possibly, in the coated vesicles which are more numerous in the paranuclear area. EM studies have confirmed this hypothesis by revealing the presence of AP in the granules and in the coated vesicles in addition to the perinuclear cisterna and

some strands of the RER. By contrast AP is not detected in the Golgi apparatus, a finding that is of particular relevance to the understanding of the mechanism of the granulogenesis (Figs. 2 and 4).

FIGURE 2 Cytochemistry of human peripheral blood LGL. a) Staining for ANAE activity reveals a paranuclear accumulation of the reaction product. b) Staining for AP with a Gomori-type of substrate shows granular deposits of the reaction product in the majority of the cells. c) Ultrastructural localization of AP activity within granules and coated vesicles (arrows). d) Ultrastructural localization of TPP in a portion of a LGL. The reaction product is found within the perinuclear cisterna, the stacks of Golgi-tubules and the Golgi-derived vesicles. Granules, as well as coated vesicles are negative.

FIGURE 4 A schematic representation of the sequence which may lead to granule formation in LGL. This sequence has been deduced by ultrastructural and cytochemical analysis of LGL from normal bone marrow or from patients with abnormally expanded LGL populations. a) Smooth vesicles (GDV) budding from the Golgi apparatus (G). b) Coated vesicles (CV) derived from the rough endoplasmic reticulum (RER). c) Coated vesicles (CV) fusing with the smooth vesicles to form multivesicular bodies (MVB). d) Multivesicular bodies showing initial deposition of an electron-dense matrix (MVB) and maturing granules (MG).

Although identified as lysosomes, the granules or the coated vesicles never display peroxidase activity in contrast to that observed for cells of the granulocytic-monocytic lineage (Ferrarini et al., 1980). In addition, both the granules and the vesicles are not involved in the process of phagocytosis, since LGL fail to ingest latex particles or opsonized red cells (Grossi et al., 1982).

PATTERNS OF GRANULE FORMATION IN LGL

In principle, the coated vesicles and the electron dense granules of LGL could represent distinct lysosomal types. Alternatively, the coated vesicles could subserve the function of transporting acid hydrolases from the RER to the mature granules. The latter hypothesis seems supported by the observations that coated vesicles fusing with immature granules (i.e., organelles containing a scarce matrix of low electron-opacity) are sometimes observed in mature LGL. This fusion occurs generally in the proximity of the Golgi apparatus where the immature granules and the coated vesicles are also more numerous indicating that the Golgi apparatus may be the site where the granules are packaged (Grossi et al., 1982).

The opportunity of investigating further the mechanism of granule formation has been offered by the availability of immature LGL populations. These were the cells from a patient with an abnormally expanded LGL population, possibly of lalignant origin (Ferrarini et al., submitted) (Fig. 3) and the LGL isolated from normal (i.e., nonneoplastic) bone marrow aspirates (Grossi et al., 1981). Cells from both sources had very few mature granules and displayed a number of features indicating active granulogenesis such as: 1) an expanded Golgi apparatus budding numerous smooth vesicles; 2) some AP-positive coated vesicles, fusing with the smooth Golgi-derived vesicles to form 3) AP-positive multivesicular bodies containing coated vesicles surrounded by the Golgi-derived smooth membranes; 4) multivesicular bodies developing into granules through the accumulation of an electron-dense matrix. That the smooth vesicles, forming the limiting membrane of the multivesicular bodies were Golgi-derived, was shown by their positivity for a specific marker of the Golgi membranes, namely, thiamine-pyrophosphatase (TPP) (unpublished observations) (Fig. 2).

FIGURE 3 Ultrastructure of a LGL from a patient with an abnormally expanded LGL population. It is of note that numerous multivesicular bodies (arrow) were found in the cytoplasm of these cells. These organelles are rarely seen in normal peripheral blood LGL.

Taken together, these observations suggest that acid hydrolases, synthesized within the RER, are transported by coated vesicles to the proximity of the Golgi apparatus where the coated vesicles fuse with Golgi-derived smooth vesicles to form multivesicular bodies which are the precursors of mature granules (Ferrarini et al., submitted) (Fig. 4).

CONCLUSIONS

Morphology and cytochemistry provide unique criteria for LGL identification, since other phenotypic characteristics so far investigated, such as the classical membrane markers or antigenic specificities detectable by monoclonal reagents, seem largely shared with cells of toher lineages (Reinherz et al., 1980; Abo and Balch, 1981; Ortaldo et al., in press). A possible exception is represented by the recently described HNK-1 monoclonal marker (Abo and Balch, 1981). The prominent morphological and cytochemical feature of LGL is constituted by a lysosomal system with different properties from those of other hemic granular cells (i.e., granulocytes and monocytes). The lysosomal system of LGL is clearly nonoperating in phagocytic functions. Since LGL possess cytotoxic activities, the possibility should be considered that the granules participate in the mechanism of killing, as would be supported by two observations, namely: 1) although capable of binding to the target, immature LGL equipped with a low number of mature granules are inefficient in killing (Ferrarini et al., submitted); 2) Chediak-Higashi patients have an impaired NK function (Haliotis et al., 1980) and their LGL display the same type of lysosomal defect detected in other cells (Abo, Balch, and Cooper, 1981, personal communication).

Granule formation does not normally occur in peripheral blood LGL but is observed in expanded, possibly malignant, LGL populations or in normal bone marrow cells. Since in other hemic lineages, granulogenesis takes place during cell maturation (Cohn and Benson, 1965; Bainton and Farquhar, 1966), it is likely that this is also the case of LGL. Granulogenesis might, therefore, represent a marker of LGL maturation.

REFERENCES

Abo, T., Balch, C.M. J. Immunol. 1981; 127:1024.

Bainton, D.F., Benson, B. J. Exp. Med. 1965; 121:135.

Costello, C., Catovsky, D., O’Brien, M., Morilla, R., Varardi, S. Leuk. Res. 1980; 5:463.

Ferrarini, M., Cadoni, A., Franzi, A.T., Ghigliotti, C., Leprini, A., Zicca, A., Grossi, C.E. Eur. J. Immunol. 1980; 10:562.

Ferrarini, M., Romagnani, S., Montesoro, E., Zicca, A., Del Prete, G. F., Nocera, A., Maggi, E., Leprini, A., and Grossi, C. E. (1981) submitted.

Grossi, C.E., Webb, S.R., Zicca, A., Lydyard, P.M., Moretta, L., Mingari, M.C., Cooper, M.D. J. Exp. Med. 1978; 147:1405.

Grossi, C.E., Burgio, V.L., Ferrarini, M. Haematologica. 1981; 66(suppl.):159.

Grossi, C.E., Cadoni, A., Zicca, A., Leprini, A., Ferrarini, M. Bloo. 1982; [in press.].

Haliotis, R., Roder, J., Klein, M., Ortaldo, J.R., Fauci, A.S., Herberman, R.B. J. Exp. Med. 1980; 151:1039.

Ortaldo, J.R., Sharrow, S.O., Timonen, T., Herberman, R.B. J. Immunol. 1981; [in press.].

Pappenheim, A., Ferrata, A. Folia Haemat. 1911; 10:78.

Payne, C.M., Glasser, M. Blood. 1981; 57:567.

Reinherz, E.L., Moretta, L., Roper, M., Breard, J.M., Mingari, M.C., Cooper, M.D., Schlossman, S.F. J. Exp. Med. 1980; 151:969.

Saksela, E., Timonen, T., Ranki, A., Hayry, P. Immunol. Rev. 1979; 44:71.

Timonen, T., Ortaldo, J.R., Herberman, R.B. J. Exp. Med. 1981; 153:569.

Winchester, R.J., Fu, S.M., Hoffman, T., Kunkel, H.G. J. Immunoo. 1975; 114:1210.

¹Supported by grants from the Italian CNR

ANALYSIS OF NATURAL KILLER ACTIVITY OF HUMAN LARGE GRANULAR LYMPHOCYTES AT A SINGLE CELL LEVEL

Tuomo Timonen¹, John R. Ortaldo and Ronald B. Herberman,     Biological Research and Therapy Branch, National Cancer Institute Frederick, Maryland

Publisher Summary

This chapter reviews natural killer (NK) activity of human large granular lymphocytes (LGLs) at a single cell level. LGLs comprise approximately 10% of peripheral blood lymphoid cells, and they can be enriched up to ≥ 90% purity by discontinuous density gradient centrifugation and subsequent depletion of high-affinity sheep erythrocyte rosette-forming cells from low density, LGL-enriched populations. In a study described in the chapter, the single-cell cytotoxicity assay in agarose was used to estimate the frequency of LGL capable of binding and lysing NK-sensitive target cells. The results indicate that up to 70% of LGL are NK cells. An additional parameter that affects the lytic potential of killer cells is their recycling capacity. It was demonstrated in another study described in the chapter, by using density gradient-purified LGL-target cell conjugates, that recycling is involved in NK lysis. It was shown that interferon can augment all of the above mentioned phases of NK cell-mediated cytotoxicity. The data obtained do not indicate that LGL are a homogenous subpopulation of lymphoid cells. On the contrary, about 20–30% of them have no detectable NK activity, there is a clear size and density heterogeneity among LGL, and they are antigenically heterogeneous.

INTRODUCTION

Recent evidence has shown an association of human natural killer (NK) activity with a morphological subpopulation of peripheral blood mononuclear cells, called large granular lymphocytes (LGL) (Timonen et al., 1979; Timonen et al., 1981). LGL comprise approximately 10% of peripheral blood lymphoid cells and they can be enriched up to 90% purity by discontinuous density gradient centrifugation and subsequent depletion of high affinity sheep erythrocyte (E) rosette-forming cells from low density, LGL-enriched populations (Timonen et al., 1981). The availability of this purified population has facilitated analyses of ultrastructure (Carpen et al., 1982), surface antigenic phenotype (Ortaldo et al., 1981), proliferative capacity (Timonen et al., submitted for publication), lytic machinery (Carpen et al., 1981) and target cell selectivity (de Landazuri et al., 1981) of NK cells. Although most, if not all, peripheral blood NK activity is exerted by LGL, little information exists as to what proportion of LGL can actually function as NK cells. This question is directly relevant to the reliability of LGL morphology as a marker for NK cells. We have used the single cell cytotoxicity assay in agarose (Grimm et al., 1979) to estimate the frequency of LGL capable of binding and lysing NK-sensitive target cells. The results indicate that up to 70% of LGL are NK cells. An additional parameter which affects the lytic potential of killer cells is their recycling capacity. We demonstrate here, by using density gradient-purified LGL-target cell conjugates, that recycling is involved in NK lysis. Furthermore, we show that interferon (IFN) can augment all of the above mentioned phases of NK cell-mediated cytotoxicity.

Binding of LGL and Other Lymphoid Cells on K562 Target Cells and Estimation of NK Cell Frequencies Among LGL

The evaluation of the frequency of killer cells among lymphoid cells has recently been facilitated by the development of the single cell cytotoxicity assay in agarose (Grimm et al., 1979). When various fractions, obtained by discontinuous density gradient centrifugation, were tested for NK cell frequencies by this method, using K562 (the cell line derived from a patient with chronic myelogenous leukemia) as a target cell, the following was seen (Table 1): a) the distribution of LGL and NK cells is similar, b) the number of LGL in each fraction is higher than the number of NK cells, c) among the purest LGL populations (fractions 2 and 3), only LGL bound to the target cells and a high cytotoxic activity was detected, d) among pure non-LGL populations (fractions 6 and 7), some lymphocytes bound to the target cells, but no cytotoxicity could be detected, e) preincubation of effector cells with IFN increased the number of NK cells among binding cells, but the number of killer cells never exceeded the frequency of LGL among binding cells. Taken together, the data indicate that LGL are the principal NK cells in peripheral blood. They also suggest that not all LGL are NK cells, since only about half of them bound to K562 after a single centrifugation of effector cells and target cells together. However, it was possible that the conjugation assay was not sensitive enough to detect all the conjugate-forming cells, or that for some reason, not all LGL are capable of binding target cells simultaneously. To test these possibilities, LGL purified by discontinuous density gradient centrifugation (fractions 2 and 3) and subsequent depletion of high affinity E-rosette forming cells, and target cells (either K562 or the anchorage-dependent mammary carcinoma line G-11) were centrifuged together at a 1:1 ratio, and the resulting conjugates and unbound target cells were separated from the rest of LGL by one step discontinuous density gradient centrifugation on 10% Percoll (Timonen et al., 1979; Carpen et al., 1981). The unbound LGL were then tested for their conjugation capacity with fresh target cells (Table 2). Some of the remaining LGL were shown to be capable of binding and lysing K562 and G-11. Even after a second depletion of conjugate-forming cells some unbound LGL were lytic. Thus, either the sensitivity of the binding assay is not sufficient to detect all the binders simultaneously, or LGL are not capable of synchronized binding. When the cumulative frequency of NK cells cytotoxic against K562 was calculated from the sequential conjugation experiments, about 50% of non-IFN-pretreated LGL and about 70% of IFN-pretreated LGL were shown to be NK cells. It is of interest that the number of NK cells against the anchorage-dependent G-11 was much less than against K562, and that IFN could increase the number of binding cells against G-11, whereas the number of binding cells did not change in the K562 system as a result of IFN-pretreatment of LGL. We have shown elsewhere that this dissociation between anchorage-dependent and suspension grown target cells is detectable by using various other target cells also (Carpen et al., 1981). The results suggest that there is some target cell selectivity in the binding capacity of LGL, and that IFN may augment NK activity by different mechanisms.

Table 1

Binding and Lytic Characteristics of Human Peripheral Blood Lymphoid Cells Fractionated by Discontinuous Density Gradient Centrifugation on Percoll

a)Adjusted to 285 mOsm/kg H2O(Timonen et al., 1981).

b)In 4 hour chromium⁵¹-release assay.

c)Analyzed by a 12 hour single cell cytotoxicity assay. (Grimm et al., 1979). A 12 hour assay was used. Cells were preincubated for 3 hours in medium (−IFN) or with 800 IU/ml of fibroblast IFN (+IFN) (Timonen et al., 1982).

Table 2

Binding and Killing of K562 and G-11 byInitially Nonadherent LGL After Removal of Primary LGL-Target Cell Conjugatesa)

a)Conjugates, produced by mixing effector cells and target cells at 1:1 ratio, were centrifuged at 120g for 10 minutes. After a gentle resuspension, the conjugates were layered on a 10% Percoll gradient and spun at 40g for 8 minutes. Target-nonadherent cells were tested for conjugation capacity against fresh target cells (Binding cycles II and III) (Timonen et al., 1979; Timonen et al., 1982).

The Effect of Neuraminidase on the Binding and Lytic Capacities of LGL

Regardless of whether the detected partial binding capacity of LGL is due to de novo activation, reactivation, maturation, or low affinity binding capacity of some LGL, sialic acid appears to be involved in the refractory, nonbinding state of LGL. If LGL and/or K562 were pretreated with 1 IU/ml of neuraminidase (VCN, Behringwerke AG, Marburg, Germany), increased binding and increased percentage of lytic cells among binders were detected (Table 3). The results indicate that practically all LGL estimated by sequential adsorption experiments to possess lytic potential could be induced to lyse simultaneously. This suggests that at least the insensitivity of the binding assay is not the major reason for the incomplete detection of NK cells among LGL by a single centrifugation of effector cells and target cells.

Table 3

The Effect of Neuraminidase (VCN) Treatment on the Binding and Lytic Capacity of LGLa)

a)K562 and/or LGL were pretreated for 1 hour at 37°C in serum-free conditions with 1 U/ml VCN, washed, and used in the single cell cytotoxicity assay. %Cytotoxicity measured with a 4 hour chromium⁵¹-release assay.

Recycling

A further dimension in the lytic capacity of lymphoid cells, in addition to binding and subsequent induction of lytic machinery, is their ability to sequentially lyse several target cells. This phenomenon, called recycling, has been suggested by an indirect method of analysis, to be involved also in NK activity (Ullberg et al., 1981). We have directly studied the recycling capacity of purified LGL, by isolating LGL-K562 conjugates by one-step discontinuous density gradient centrifugation on Percoll as in Table 2. Both the rate of spontaneous dissociation of the conjugates and the capacity of the dissociated LGL to rebind K562 were then measured, along with the single cell cytotoxicity assay in agarose for the detection of NK cells among reconjugated LGL. As shown in Table 4, the majority of LGL spontaneously dissociated rapidly after the initial conjugate formation. If the LGL were not pretreated with IFN, there was a refractory period, and then rebinding approximately after 120 min. The results from the single cell assay indicate that the recycling is mainly regulated at the binding level, since during the whole experiment all those LGL that bound target cells lysed them with a similar efficiency. If the effector cells were pretreated with IFN, the rebinding and lysis could take place continuously, indicating that IFN can increase the recycling capacity of NK cells.

Table 4

Lytic Capacity of Recycling LGLa)

a)Enriched LGL (after preincubation with or without IFN) -K562 conjugates were incubated for various time periods at 37°C and the spontaneous dissociation (without centrifugation) or the rebinding capacity after centrifugation at 120g for 10 minutes (with centrifugation) were monitored.

CONCLUSIONS

The morphological and density analyses of the NK cells in human peripheral blood strongly suggest that most, if not all, NK cells are morphologically LGL. Furthermore, we conclude that there is a high percentage of actual NK cells among LGL. Although other groups have reported NK activity in lymphoid populations apparently devoid of LGL (Hokland et al., 1981), we have not been able to do so, even by using numerous other target cells in addition to K562 (Carpen et al., 1981, de Landazuri et al., 1981).

Our data do not indicate that LGL are a homogenous subpopulation of lymphoid cells. On the contrary, about 20-30% of them have no detectable NK activity, there is a clear size and density heterogeneity among LGL, and they are antigenically heterogenous (Ortaldo et al., 1981). It is possible that the LGL inactive against K562 are cytotoxic to another target cell, since polyclonality of NK cells has been suggested (Phillips et al., 1980). However, using seven different target cells we have shown that polyclonality can maximally account for an additional 10% LGL as NK cells, i.e., NK cell frequencies among LGL appear to be maximally around 80%. It is of interest that LGL reacting with the monoclonal antibody OKT8 do not seem to account for an appreciable proportion of NK activity (Ortaldo, et al., 1981; Timonen et al., submitted for publication). It will be important to study whether OKT8+ LGL would be K cells responsible for antibody-dependent cell-mediated cytotoxicity, some immature forms of NK cells not readily inducible to NK activity with IFN, or totally unrelated to NK activity, maybe exerting the suppressor cell activity known to be associated with the reactivity with OKT8 (Reinherz et al., 1980).

The net result detected in the 51chromium-release assays from the cytolytic activity of LGL can be attributed to the binding, lytic and recycling capacities of LGL. Therefore, mere calculation of the frequency of LGL among effector cells is not a sufficient measure of NK activity, since individual variations in these three parameters occur (Ullberg et al., 1981; our published results). However, the analyses of LGL frequencies among lymphoid cell populations should be helpful in deciding whether altered NK activity is due to major changes in the frequency of LGL, or to altered function.

Although most of the NK activity among fresh unprimed lymphocytes is exerted by LGL, not much is known about the cellular origin of NK-like effector cells that are induced during antigenic stimulation, for example in mixed lymphocyte cultures (Seeley et al., 1980), or in fetal calf serum-containing cultures (Ortaldo et al., 1980). The availability of the purified populations of both LGL and conventional T-cells will certainly facilitate the analysis of the cellular basis of these, as well as other forms of immune response.

ACKNOWLEDGMENT

This work has been supported in part by Grant No. 1 R01 CA 23809-01 from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

REFERENCES

Carpen, O., Virtanen, I., Saksela, E. Cell Immunol. 1981; 58:97.

Carpen, O., Virtanen, I. and Saksela, E. (1982). J Immunol, in press.

Grimm, E., Bonavida, B. J Immunol. 1979; 123:2861.

Hokland, M., Heron, I., Berg, K. Antiviral Research Abstr 1. 1981; 1:83.

de Landazuri, M.O., Lopez-Botet, M., Timonen, T., Ortaldo, J.R., Herberman, R.B. J Immunol. 1981; 127:1380.

Ortaldo, J., Herberman, R.B.Herberman R.B., ed. Natural cell-mediated immunity against tumors. Academic Press: New York, 1980; 465.

Ortaldo, J.R., Sharrow, S.O., Timonen, T., Herberman, R.B. J Immunol. 1981; 127:2401.

Phillips, W.H., Ortaldo, J.R., Herberman, R.B. J Immunol. 1980; 125:2322.

Reinherz, E.L., Kung, P.C., Goldstein, G., Schlossman, S.F. J Immunol. 1980; 124:1301.

Seeley, J.K., Karre, K.Herberman R.B., ed. "Natural cell-mediated immunity against tumors. Academic Press: New York, 1980; 477.

Timonen, T., Ranki, A., Saksela, E., Hayry, P. Cell Immunol. 1979; 48:121.

Timonen, T., Ortaldo, J.R., Herberman, R.B. J Exp Med. 1981; 153:569.

Timonen, T., Ortaldo, J.R., Stadler, B.M., Bonnard, G.D., and Herberman, R.B. (1982) Submitted for publication.

Ullberg, M., Jondal, M. J Exp Med. 1981; 153:615.

¹Present address: Department of Pathology, University of Helsinki, Finland.

2

OTHER SPECIES

IDENTIFICATION AND CHARACTERIZATION OF THE NATURAL KILLER (NK) CELLS IN RATS

Craig W. Reynolds, Robert Rees¹, Tuomo Timonen² and Ronald B. Herberman,     Biological Research and Therapy Branch National Cancer Institute Frederick, Maryland

Publisher Summary

This chapter discusses identification and characterization of the natural killer (NK) cells in rats. Recent studies have suggested that NK cells are important mediators of a number of in vitro immunological phenomena. In a study described in the chapter, all experiments were performed with Wistar–Furth (WF) or Fischer (F344) rats. The rat G1-TC, the mouse YAC-1, and the human K562 leukemias were maintained in vitro as suspension cultures. The rat cell lines that were maintained in vitro as monolayer cultures were W/Fu-T and ErTH/V-G rat sarcomas, the R35 rat mammary adenocarcinoma, and the F2304 rat embryo cell. Spleen, thymus, lymph node, bone marrow (BM), peritoneal exudate cells (PEC), and peripheral blood leukocytes (PBL) were prepared in Hanks’ balanced salt solution (HBSS). The removal of adherent cells was done by incubating leukocyte preparations on columns of nylon–wool (NW) at 37°C. It was found that there are cells in rat peripheral blood with the morphological features of large granular lymphocytes (LGL). Small lymphocytes and a monocyte were also seen, demonstrating the intermediate size of rat LGL.

I INTRODUCTION

Recent evidence has suggested that natural killer (NK) cells are important mediators of a number of invitro immunological phenomena. Little evidence, however, has been produced that directly examines the role of the NK cell in the invivo mediation of these functions. A major reason for this deficiency is the lack of a suitable animal model that would permit the purification, transfer, and direct invivo testing of NK cell function. Recently, however, we have been able to identify and isolate NK cells from rat spleen and blood (Reynolds et al., 1981a; Reynolds et al., 1981b). The present manuscript will review the morphology, organ distribution, frequency and target specificity of these rat LGL. The findings suggest that rats may provide a very useful animal model for detailed studies on the ontogeny, regulation and invivo relevance of NK cells.

II MATERIALS AND METHODS

Animals

All experiments were performed with Wistar-Furth (WF) or Fischer (F344) rats.

Target Cells

The rat G1-TC, the mouse YAC-1 and the human K562 leukemias were maintained invitro as suspension cultures (Oehler et al, 1978). In addition, the following rat cell lines were maintained invitro as monolayer cultures: W/Fu-T and ErTH/V-G rat sarcomas, the R35 rat mammary adenocarcinoma and the F2304 rat embryo cell (Nunn et al., 1976).

Preparation of Lymphoid Cells

Spleen, thymus, lymph node, bone marrow (BM), peritoneal exudate cells (PEC) and peripheral blood leukocytes (PBL) were prepared in Hanks’ balanced salt solution (HBSS) as previously described (Ortiz de Landazuri & Herberman, 1972; Oehler et al., 1977). The removal of adherent cells was done by incubating leukocyte preparations on columns of nylon-wool (NW) at 37° C.

Percoll Fractionation - Evaluation of Cell Morphology

NW-nonadherent lymphocyte preparations were separated by centrifugation on discontinuous density gradients of Percoll (Pharmacia Chemicals, Uppsala, Sweden), as previously described (Reynolds et al, 1981a). For the morphological evaluation of lymphocyte preparations, air-dried cytocentrafuge preparations were fixed in methanol and stained with 10% Giemsa (pH 7.4).

⁵¹Cr-release Cytoxicity Assay

Two-fold serial dilutions of effector cells were mixed with 51Cr-labeled target cells and incubated at 37° C for 4 hours, as previously described (Reynolds & Herberman, 1981). Results are expressed as either percent cytotoxicity or as lytic units (LU) per 107 effector cells (Herberman et al, In Press).

III RESULTS AND DISCUSSION

Figure 1Ademonstrates that there are cells in rat peripheral blood with the morphological features of LGL. These cells have previously been described in the human (Timonen et al, 1979a; Timonen et al, 1979b; Timonen et al, 1981; Timonen and Saksela, 1980; Saksela et al, 1979) and characteristically have a high cytoplasmic to nuclear ratio, azurophilic granules in the cytoplasm and, generally, a kidney-shaped nucleus. Small lymphocytes and a monocyte can also be seen in Figure 1A, demonstrating the intermediate size of rat LGL. The importance of LGL in NK activity was first suggested by their binding to NK sensitive target cells (Figure 1B). Subsequent studies have clearly demonstrated that these lymphocytes are also capable of killing these tumor cells (Reynolds et al, 1981a). Discontinuous density gradients of Percoll can also be used to isolate LGL and T cells from nylon wool passed blood. The morphology of these isolated populations can be seen in Figure 1C-D.

FIGURE 1 Morphology of rat peripheral blood lymphoid cells: A = Ficoll-Hypaque separated population (LGL indicated by arrow). B = LGL conjugates with G1-TC target cell. C = Percoll isolated LGL population (>80% LGL). D = Percoll isolated small lymphocytes (<5% LGL).

To determine if other organs contained LGL we screened a number of sites which had previously been shown to contain NK activity (Oehler et al, 1978; Nunn et al, 1976; Zoller & Matzku, 1980). The results in Table 1 clearly show that LGL are not confined to the blood. These cells are also found in significant numbers in the spleen, peritoneal cavity, and lungs. A small but significant number of LGL was always observed in lymph nodes but few, if any, LGL were found in the thymus and bone marrow. In addition, the passage of leukocyte preparations over nylon-wool columns always enriched for LGL but rarely enriched for NK activity. With the exception of the lungs, the LGL frequency pattern from various organs was similar to the distribution of NK activity (PBL > spleen > peritoneal exudate > lymph node > thymus = bone marrow). Interestingly, even the preincubation of lung-LGL with interferon (IFN) preparations did not greatly augment their cytotoxic potential (data not shown). These results are in general agreement with the hypothesis that rat NK cells are LGL, but also demonstrate that not all LGL have cytolytic activity. Mere measurement of LGL frequency would not appear to be an adequate means for predicting the NK activity of an effector cell population.

Table 1

LGL Frequency and Organ Distribution of NK Activity in W/Fu Rats

a E/T % 100:1 with G1− TC target.

To directly examine the association of rat LGL with NK activity, Percoll density gradients were run using nylon-wool passed lymphocyte preparations (Figure 2).

FIGURE 2 = % of LGL.

These experiments demonstrated that most of the NK activity in both spleen and blood was contained in a minor population of cells which consisted primarily of LGL. These cells were highly enriched in fractions 2 and 3 with purities of 80-95% obtainable from the blood and 60-80% from the spleen. The enrichment of NK activity in the LGL fractions, the absence of NK in the LGL-depleted fractions, and the binding and lysis of NK susceptible target cells (data not shown) strongly indicate that rat LGL are the NK effector cell in this species.

Since the data in Figure 2 was obtained using a single lymphoma target (YAC-1), we were interested to know whether the cytotoxic cell for other targets was also an LGL. The results in Figure 3 demonstrated an enrichment in NK activity in fraction 2/3 with a corresponding decrease in both LGL and NK activity in later fractions. These results suggest that the natural cytotoxicity which is seen against syngeneic lymphomas (G1-TC) syngeneic embryo cells (F2304), allogeneic sarcomas (ErTH/V-G, W/FU-T) and adenocarcinomas (R35), and xenogeneic leukemias (YAC-1, K562) is primarily mediated by LGL. These data imply that any heterogeneity in natural effectors for lymphoma targets and monolayer targets that may exist is within the small LGL subset.

FIGURE 3 = K562. E/T ratio = 50:1. ( ) = Average % LGL in each fraction.

REFERENCES

Herberman, R. B., Ortaldo, J. R., and Timonen, T. Methods in Enzymol (in press).

Nunn, M.E., et al. J. Natl. Cancer Inst. 1976; 56:393.

Oehler, J.R., et al. Cell Immunol. 1977; 29:238.

Oehler, J.R., et al. Int. J. Cancer. 1978; 21:204.

Ortiz de Landazuri, M., Herberman, R.B. J. Natl. Cancer Inst. 1972; 49:147.

Reynolds, C.W., Timonen, T., Herberman, R.B. J. Immunol. 1981; 127:282.

Reynolds, C.W., et al. J. Immunol. 1981; 127:2204.

Reynolds, C.W., Herberman, R.B. J. Immunol. 1981; 126:1581.

Saksela, E., et al. Immunol. Rev. 1979; 44:71.

Timonen, T., et al. Cell Immunol. 1979; 48:121.

Timonen, T., et al. Cell Immunol. 1979; 48:133.

Timonen, T., Saksela, E. J. Immunol. Methods. 1980; 36:285.

Timonen, T., Ortaldo, J.R., Herberman, R.B. J. Exp. Med. 1981; 153:569.

Zoller, M., Matzku, S. J. Immunol. 1980; 124:1683.

¹Present address: Department of Virology, University of Sheffield Medical School, Sheffield, England.

²Present address: Department of Pathology, University of Helsinki Central Hospital, Helsinki, Finland.

LARGE GRANULAR LYMPHOCYTES AS EFFECTOR CELLS OF NATURAL KILLER ACTIVITY IN THE MOUSE

Aldo Tagliabue¹, Diana Boraschi¹, Saverio Alberti² and Walter Luini²,     ¹Sclavo Research Center, Siena, Italy; ²IRFMN, Milan, Italy

Publisher Summary

This chapter reviews large granular lymphocytes (LGL) as effector cells of natural killer (NK) activity in the mouse. The possibility of identifying the effector cells of NK cytotoxicity by their morphology is a relatively new acquisition. In a study described in the chapter, the possible relationship between LGL and mouse NK activity was investigated. Two main approaches were used: (1) the distribution of LGL in different lymphoid and nonlymphoid organs was studied with a focus on mouse peripheral blood in analogy with the human studies and (2) this study also investigated the hypothesis that the large lymphocytes with cytoplasmatic granules, which are known to be present in high proportions in the epithelium of the small intestine of mammals, might exert NK activity at that site. This chapter summarizes the results obtained, indicating that LGL are also effector cells of NK activity in mice. Several lines of evidence suggest that NK cells are a heterogeneous population. The serological characterization of the NK effector cells in the mouse small intestine, where the cytotoxicity is mainly exerted by LGL, revealed strong similarities between these cells and the NKT subset.

I INTRODUCTION

The possibility of identifying the effector cells of natural killer (NK) cytotoxicity by their morphology is a relatively new acquisition. In fact, it is only in the last two years that a few studies have shown that lymphocytes with a high cytoplasmic:nuclear ratio and characteristic azurophilic granules in the cytoplasm (large granular lymphocytes, LGL) are associated with the in vitro expression of NK activity in humans (1, 2). We have recently investigated the possible relationship between LGL and mouse NK activity. Two main approaches were used: a) the distribution of LGL in different lymphoid and non-lymphoid organs was studied (3), with a focus on mouse peripheral blood in analogy with the human studies. b) We investigated the hypothesis that the large lymphocytes with cytoplasmatic granules, which are known to be present in high proportions in the epithelium of the small intestine of mammals, might exert NK activity at that site (4). Here we will summarize the results obtained, indicating that LGL are also effector cells of NK activity in mice, and we will report our most recent observations about murine LGL.

II EXPERIMENTAL PROCEDURES

NK activity was assessed by a 6h 51Cr release assay employing susceptible tumor target cells such as YAC-1 lymphoma (3, 4) or non-susceptible tumor targets such as MN/MCA-1 fibrosarcoma (4). Lymphoid cell populations were obtained by procedures previously described in detail (3, 4, 5). LGL were stained after cytocentrifugation with Diff-Quik (Harleco, Gibbstown, NJ). The antisera employed were: anti-Thy 1.2 from New England Nuclear (Boston, Mass.), Lyt 1.1 and Lyt 2.1 from Cedarlane Laboratories (Hornby, Canada), anti-NK 1.2 from Dr. R.C. Burton, Harvard Medical School, Mass., anti-asialo GM 1 from Dr. K. Okumura, Tokyo University. The complement dependent cytotoxic test (5) was performed employing low toxicity rabbit complement (Cedarlane Lab.).

III RELATIONSHIP BETWEEN LGL AND NK ACTIVITY

Lymphoid cell populations from different organs were first examined to determine the frequency of LGL (Table I). LGL were always detectable in organs positive for NK activity (3), whereas no LGL were found where NK activity was absent. The positive correlation between % LGL and NK activity was further demonstrated in studies with lymphocytes from the peripheral blood of mice of different ages and strains. The highest % LGL were observed in C3H/HeN mice at 4-8 weeks, i.e. at the peak of the NK activity. Moreover, nude BALB/c mice had higher % LGL and NK activity when compared to normal littermates. In addition, it was possible to obtain lymphoid populations enriched in LGL by separation of mononuclear cells from peripheral blood on discontinuous Percoll density gradients devised for mouse cells (3). In fact, in the fraction of the gradient containing 55% of Percoll (fraction 55), it was possible to obtain 40-85% of LGL when starting from an input population which contained 5-10% LGL. Table II shows a representative experiment which indicates how an increase in LGL in fraction 55 was parallel to an increase in NK activity. Interestingly, the fraction enriched in LGL was more cytotoxic against YAC-1 cells, but still failed to lyse fibrosarcoma cells, previously shown not to be susceptible to splenic NK activity (4).

Table I

Comparison between LGL and NK activity

¹in C3H/HeN mice of 8 weeks of age

²tested employing peripheral blood lymphocytes

³tested employing peripheral blood lymphocytes from C3H/HeN mice

⁴tested in a 6h ⁵¹Cr release assay against YAC-1 tumor cells.

Table II

Distribution of LGL and NK cytotoxicity among fractions of C3H/HeN peripheral blood obtained by discontinuous percoll density gradient centrifugation

aAttacker to target ratio

bP ≤ 0.05 versus input population.

IV NK ACTIVITY OF MURINE GUT MUCOSAL LYMPHOID CELLS

Lymphocytes with a morphology similar to the LGL have been known for a long time to be present in the intestinal mucosa of mammals (6). Thus, we tested purified populations of lymphoid cells from the mouse intestine for their capacity to exert NK activity. Table III shows that intraepithelial lymphocytes from the gut (IEL) had NK activity comparable to the splenocytes and that interferon (IFN-β) boosted this activity. In contrast, no cytotoxicity was observed employing lymphocytes from the Peyer’s patches with or without IFN-β. The characteristics of intestinal NK activity were similar to those of splenic NK cells (4). Intestinal NK activity was not affected by depletion of adherent cells. Moreover, the age dependency of NK activity was similar for IEL and splenocytes. Finally, NK-insensitive target cells were not lysed by IEL (4).

Table III

NK activity of lymphocytes from different organs and its boosting by IFN-β

aIncrease above spontaneous level P ≤ 0.05; for experimental details see ref. 3.

V LGL AS THE NK EFFECTOR CELL IN THE INTESTINAL MUCOSA

In an attempt to investigate whether the LGL were the NK cells in the mouse small intestine, we studied their capacity to form conjugates (2) with YAC-1 cells. As shown in Table IV, LGL were capable of preferentially forming conjugates with the tumor cells. We employed the Percoll gradients with the intestinal cells and in this case, too, it was possible to obtain populations enriched in LGL and NK activity in fraction 55, whereas fraction 70 contained very low percentages of LGL and lacked NK activity (Table IV).

Table IV

NK activity and frequency of conjugation with YAC-1 cells of intraepithelial lymphocytes fractionated on percoll gradientsa

aCells were obtained from 8 week old CBA/J mice.

VI SEROLOGICAL CHARACTERIZATION OF THE INTESTINAL NK EFFECTOR CELL

In order to better characterize the effector cells of NK activity in the murine intestinal epithelium, IEL from fraction 55 were treated with a panel of antisera and complement prior to the cytolysis assays. Table V summarizes the results obtained. It appears that the intestinal NK cell is not identical to the splenic one. In fact, the gut NK activity was greatly reduced by anti-Thy 1.2, but only marginally affected by anti-asialo-GM 1 or anti-NK 1 (5).

Table V

Effect of treatment with various antisera plus complement on NK activity of splenocytes and iel from fraction 55

aFrom 8 week old CBA/J mice

bSignificant reduction from untreated or complement controls, P≤ 0.05

cSignificant reduction from untreated or complement controls, P ≤ 0.001.

VII CONCLUSIONS AND SPECULATIONS

Taken all together, these results demonstrate that also in the mouse LGL are effector cells of NK activity. Several lines of evidence suggest that NK cells are a heterogeneous population. Thus it remains to be elucidated whether the LGL represents a subpopulation of NK cells. Interestingly, the serological characterization of the NK effector cells in the mouse small intestine, where the cytotoxicity is mainly exerted by LGL, revealed strong similarities between these cells and the NKT subset recently described by Minato et al. (7). The possibility of purifying LGL with Percoll gradients also from other organs will allow a further analysis of the NK subsets.

Lymphocytes with cytoplasmatic granules have now been described in the epithelium of other organs besides the intestine, such as the mouse lung (3), the rabbit oviductal fimbria and endocervices (8), the male reproductive tract of rats and monkeys (9). Thus, it is tempting to speculate that LGL play a major role in immunosurveillance at the mucosal level.

ACKNOWLEDGEMENTS

We would like to acknowledge the collaboration of Prof. J. Bienenstock and Drs. A.D. Befus and D.A. Clark of the McMaster University, Hamilton, Ontario where part of this study was performed by A. Tagliabue, supported by a fellowship of the "Associazione Italiana per 1a Ricerca sul Cancro„, Milan, Italy.

REFERENCES

1. Timonen, T., Saksela, E., Ranki, A., Häyry, P. Cell. Immunol. 1979; 48:133.

2. Timonen, T., Ortaldo, J.R., Herberman, R.B. J. Exp. Med. 1981; 153:569.

3. Luini, W., Boraschi, D., Alberti, S., Aleotti, A., Tagliabue, A. Immunol. 1981; 43:663.

4. Tagliabue, A., Luini, W., Soldateschi, D., Boraschi, D. Eur. J. Immunol. 1981; 11:919.

5. Tagliabue, A., Befus, A.D., Clark, D.A., and Bienenstock, J., submitted for publication.

6. Rudzik, O., Bienenstock, J. Lab. Invest. 1974; 30:260.

7. Minato, N., Reid, L., Bloom, B.R. J. Exp. Med. 1981; 154:750.

8. Odor, D.L. Fertil, and Steril. 1974; 25:1047.

9. Dym, M., Romrell, L.J. J. Reprod. Fert. 1975; 42:1.

B

CHARACTERIZATION BY SURFACE MARKERS

1

HUMAN

CHARACTERIZATION OF HUMAN NK CELLS IDENTIFIED BY THE MONOCLONAL HNK-1 (Leu-7) ANTIBODY¹

Toru Abo, Max D. Cooper and Charles M. Balch,     Cellular Immunobiology Unit of the Tumor Institute, Departments of Surgery, Microbiology, and Pediatrics, University of Alabama in Birmingham

Publisher Summary

This chapter reviews the characterization of human natural killer (NK) cells identified by the monoclonal HNK-1 (Leu-7) antibody. The monoclonal antibody, HNK-1, was produced against the cultured cell line, HSB-2, using the hybridoma technique introduced by Köhler and Milstein. Although HSB-2 cells have been considered to be of T-cell origin, they lack T-cell-associated antigens identified by the monoclonal antibodies, OKT1, T3, T4, T5, and T8. In a study described in the chapter, when the HNK-1 antibody was tested against cultured lymphoid cell lines, it reacted with some T-cell lines (HSB-2 and MOLT-4) but not with any other T-cell line (MOLT-3), with any B-cell lines, or with cultured phagocytic cells. When human blood mononuclear cells were examined by immunofluorescence, the HNK-1 antibody reacted solely with a morphologically distinct population of granular lymphocytes with NK- and K-cell function. The results suggested that a differentiation antigen identified by the monoclonal HNK-1 antibody is exclusively expressed on human NK and K cells. The HNK-1 antibody also distinguishes the classically defined NK cell from its analogues.

Natural killer (NK) cells and antibody-dependent killer (K) cells have beendefined primarily by their functional properties (1, 2). Partial purification of NK cells from lymphocyte populations has beenpossible because of their relatively lower density (3). While several types of surface markers have been identified on human NK cells, none have been precise markers for these cells because their distribution has not been restricted to NK cells (4, 5).

We have recently produced a monoclonal antibody, HNK-1, that reacts with a differentiation antigen on human NK and K cells (5–8). We describe herein some characteristics of human NK cells identified by the HNK-1 (Leu-7) antibody, and suggest a possible differentiation scheme for NK cells.

I PRODUCTION OF THE HNK-1 MONOCLONAL ANTIBODY

The monoclonal antibody, HNK-1, was produced against the cultured cell line, HSB-2, using the hybridoma technique introduced by KÖhler and Milstein (5). Although HSB-2 cells have been considered to be of T cell origin, they lack T cell-associated antigens identified by the monoclonal antibodies, OKT1, T3, T4, T5 and T8 (5, 9). In an earlier study, Kaplan et al. reported that a rabbit antiserum produced against HSB-2 reactedwith human NK and K cells as well as with T cells (10).

When the HNK-1 antibody was tested against cultured lymphoid cell lines, it reacted with some T cell lines (HSB-2 and MOLT-4) but not with any other T cell line (M0LT-3), with any B cell lines or with cultured phagocytic cells (5). When human blood mononuclear cells were examined by immunofluorescence, the HNK-1 antibody reacted solely with a morphologically distinct population of granular lymphocytes with NK and K cell function.

II NATURE OF THE HNK-1 ANTIGEN

The HNK-1 antigen was identified both on the surface membrane and in the cytoplasm of granular lymphocytes by both direct and indirect immunofluorescence (5, 6).

Although the HNK-1 antigen was exclusively expressed on functional NK and K cells, it does not appear to be a functional receptor (5). Thus, neither NK nor K cell function was decreased when mononuclear cells were pretreated with the HNK-1 antibody or when the antibody was added to the culture medium for NK and K cell functional assays. The HNK-1 antigen expression on the cell surface was also resistant to treatment with 0.1% pronase, a concentration that inhibited NK and K cell functions.

III CHARACTERISTICS OF HUMAN NK CELLS IDENTIFIED BY THE HNK-1 ANTIBODY

A Morphology and Cytotoxic Function of HNK-1+ Cells

The HNK-1 antibody reacted with a minority of peripheral blood lymphocytes (15 ± 7%) from normal adult donors (5). The HNK-1+ cells, purified with a fluorescence-activated cell sorter, were a homogeneous population of medium-sized lymphocytes with abundant neutrophilic cytoplasm. This morphologicalappearance is similar to that previously described for NK cells isolated by direct adherence to target cells by Saksela et al. (11). On the other hand, the HNK-1− cells displayed the classical features of small lymphocytes with a narrow rim of cytoplasm and no granules.

The sorted HNK-1+ and HNK-1„ cells were also examined for NK and K cell function against K562 target cells and antibody-coated chicken erythrocytes. Almost all of the cytotoxcity resided in the HNK-1+ cell population (5).

B Expression of Other Surface Antigens on HNK-1+ Cells

In spite of homogeneity of the morphology, cytotoxic function and most surface markers, HNK-1+ cells are heterogeneous with respect to E-rosette receptor expression. HNK-1+ cells distribute in both null cell (ER−sIg−Fcγ R+) and the Tγ cell (ER+FcγR+) fractions (5).

The presence of T cell-associated antigens on HNK-1+ cells was examined further using other monoclonal antibodies and two-color immunofluorescence (7). The HNK-1+ subpopulation of cells showed variable expression of T cell antigens. The pan-T cell antigens, 3A-1, L17F12, T1 and T3, were co-expressed on 20 to 50% of HNK-1+ cells. Ten to 20% of HNK-1+ cells were reactive with T4, T5 and T8