Immunobiological and immunochemical aspects of the T-200 family of glycoproteins

~okcularhmunology, Vol. 21, No. 11, pp. 1113-1121.1984 0161-589W84 $3.00 + 0.00 Printed in Great Britain Q 1984 Pergamon Press Ltd

IMMUNOBIOLOGICAL AND IMMUNOCHEMICAL ASPECTS OF THE T-200 FAMILY OF GLYCOPROTEINS

WALTER NEWMAN,* STEPHAN R. TARCANt and LOREN D. FASTS

*Division of Immunobiology, Ortho Pharmaceutical Corporation, Raritan, NJ 08869, U.S.A.; TGeriatric Research, Education and Clinical Center, Medical Research Services, Wadsworth VA

Medical Center and Division of Gastroenterology, Department of Medicine, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.; and fDivision of Clinical Hematology,

Rhode Island Hospital, Providence, RI 02902, U.S.A.

(Accepted 10 July 1984)

Abstract-We present some new structural and functional data on the family of high mol. wt glycoproteins of human hematopoietic cells which strongly suggests they play a role proximate to certain Ca’+-dependent events. Structural data on this complex are presented which describe elements of its tertiary structure and associations between the different glycoproteins in the plasma membrane. The mol. wt profile of the T-200 structures on highly enriched natural killer (NK) cells is used to support an argument for a T-cell vs a myeloid nature for NK cells. Furthermore, certain murine monoclonal antibodies directed to a particular epitope of T-200 on NK cells are shown to block cytolysis. The specificity of this blockade strongly suggests that T-200 may be an NK receptor. A mapping of this effect has allowed us to define a new stage of the lytic cycle, termed “triggering”. This very rapid event occurs subsequent to conjugation between the NK and the target cell, and prior to the Ca’+-dependent second- phase programming for lysis. A close association between “triggering”, as defined by antibodies to T- 200, and Cazi-dependent stimulus-secretion events support the concept of T-200 as a receptor structure.

INTRODUCTION

Virtually all hematopoietic cells of the mouse, the

rat and humans express one or more members of the high mol. wt glycoprotein family known collectively as T-200, and in the mouse as defined by anti-l? 5 sera (Komuro et al., 1975; Trowbridge et al., 197% b; Omary et al., 1980; Trowbridge, 1978; Trow- bridge and Mazauskas, 1976). Murine alloantisera have distinguished two alleles, Ly 5.1 and Ly 5.2, which map to chromosome 1. On mouse cells there are three members of this group whose sizes are 200, 205 and 220K. Sarmiento et al. (1982) have recently developed a monoclonal antibody to a fourth member whose mol. wt is approximately 200,000, and which only appears after stimulation of T-cells with lectin or alloantigen. Most recently, two additional members of this family at 210 and 215K have been described on murine Ly 2, 3 T-cells (Tung et al., 1983). On human cells there are four members at 180, 190, 210 and 220K. One of the curious features of this family is that their expression is characteristic of a cell’s lineage. Hence the murine 220K structure is found predominantly on B-cells (Coffman and Weissman, 1981), while the 220K member is largely restricted to T-cells (Tung et al., 1981). With human T-200, however, this is not a hard and fast rule (Morishima et al., 1982). Resting T-cells, for example, express all four members of this group,

though B-cells and monocytes tend to express only the two higher members. Though these experiments are with uncloned cells, even T- and B-cell lines of human origin tend to express more than one

member of this family (Morishima et al., 1982; Dalchau ef al., 1980). In all these cases it is not clear whether these different mol. wt forms represent a single gene product modified post-translationally, or whether they represent three or four closely related gene products.

Watson et al. (1981) have shown that the murine members of this group, when studied for their biosynthetic characteristics, have apparently distinct precursors. More recently, Tung et al. (1984) have presented evidence for protein structural differences between the murine 200 and 220K forms. Morishima et al. (1982) have examined the carbohydrate com- position of the different members of the human T- 200 family, and found a difference in the sialic acid content of the higher and lower mol. wt bands. This difference in itself, however, is not enough to account for the total difference in mol. wt, and Tung et al. (1984) were unable to reduce the differences in mol. wt between the murine 220 and 200K forms by digestion with a variety of glycosidases. Another salient feature of these molecules is their large intracytoplasmic domains, and the fact that they are phosphorylated on a serine residue near the C- terminus (Omary and Trowbridge, 1980; Watson, 1982).

With all of this structural information it is perhaps surprising that we know so little about the function of these structures. Most speculation has centered around the possibility that they play some role in T- or B-cell differentiation, migration or interaction with other cell types (Trowbridge and Mazauskas, 1976; Coffman and Weissman, 1981; Morishima et

1113

1114 WALTER NEWMAN et al.

al., 1982). One clue from the work of Kasai et al.

(1979) [see also Komuro et al. (1975) and Trow- bridge and Mazauskas (1976)] confirmed by reports from other laboratories (Pollack et al., 1979; Minato et al., 1980; Seaman et al., 1981; Sparrow and McKenzie, 1983) is the finding that anti T-200 blocks the activity of natural killer (NK) cells.

Our interest in these cell surface antigens arose during the course of experiments designed to determine which membrane structures on human NK cells are involved in the recognition and lysis of susceptible target cells. NK cells are a population of lymphoid cells, perhaps related to T-cells, which have been postulated to play a key role in the body’s defenses against virus infection and perhaps tumori- genesis (Herberman, 1982; Hoshino et al., 1984). Unlike T-cells, however, NK cells need no prior exposure or priming to trigger cytolysis. The nature of their receptors and the target structures they recognize is largely unknown. Our approach to the receptor problem was to develop murine monoclonal antibodies which blocked the ability of human NK cells to lyse the susceptible tumor target K562. Our screening procedures were designed to detect blocking antibodies, not anti-T-200 reagents per se. However, such antibodies were developed and shown in all cases to react with a particular epitope of T-200. The experiments described later outline the analyses we performed to ascertain the specificity and mode of action of these antibodies. In addition, we provide some additional structural details on the T-200 complex, its disposition in the membrane, and the nature of the T-200 structures on highly enriched NK cells. Overall, these results suggest a central role for T-200 in transmitting, from the cell exterior to the cytoplasm, signals proximate to Ca’+-dependent stimulus-secretion coupling. The possibility that this function, termed “triggering”, may be a receptor function of NK cells, is

discussed.

MATERIALS AND METHODS

The materials and methods used for the work presented here have been presented elsewhere (Newman, 1982; Targan and Newman, 1983; New- man et al., 1983; Fast et al., 1983) with the exception of the cross-linking studies. Those were performed with the reagent dithiobis (succinimidyl propionate) (DSP) (Pierce, Rockford, IL) according to the procedure of Lutz et al. (1977), with minor modifi- cations. Briefly, 10’ cells were washed 3 times in PBS and resuspended in PBS, to which DSP was added to 0.5 mM. The reaction was carried out at 4°C for 30 min. Controls were treated in DMSO- PBS without DSP. The reaction was stopped by washing the cells, which were then iodinated as previously described for immunoprecipitation analysis (Newman et al., 1983).

RESULTS

The anti-T-200 monoclonal antibodies used in these experiments are described in Table 1. Anti- bodies 13.1 and 13.3 block NK cell function and demonstrate a 50% effect at approximately 1 ngiml. The control anti-T-200 antibodies 13.5 and 13.6 are essentially without effect. The inhibition caused by 13.1 and 13.3 is entirely a consequence of the binding of antibody to the killer cell and not to the target (Newman, 1982). Sequential immunoprecipitation data have shown that all 13.1- and 13.3- bearing T-200 molecules also bear the 13X3.6 epitope. The former pair react with and define the “A”-epitope, while the latter react with and define the “B”-epitope. These two sites are distinct as shown by competition radiobinding assays and partial-proteolysis experiments (Newman et al., 1983). Moreover, the partial-proteolysis experiments showed that the B-epitope resides between the membrane of the cell and the A-epitope, which

Table 1. Monoclonal antibodies used in this study

Designation Subclass T-200

epitope” NK

blocking” t

(m%)< Reference

13.1 IgGl

13.3 IgGl 13.4 IgG2 13.5 IgG 1 13.6 IgGl

I ng 40

1 ng 40

>l I*g nd”

>I )1g 180

>l Pg 180

Newman (1982) Newman et a/. (1983) Newman el al. (1983) Fast et al. (1983) Newman et al. (1983) Newman et al. (1983)

a Determined by competition radiobinding assays and limited-proteolysis experiments. ’ Amount required for i/2 maximal effect, in a 4-hr 51Cr release assay with K562 target cells. ’ Time required for % of the cell-bound ‘*“I-labeled antibody to dissociate in the presence of a 20-fold excess

of homologous unlabeled antibody. All experiments were performed with the U-937 tumor cells. d Not done.

T-200 family of glycoproteins 1115

is proximate to the N-terminus. The avidity of the

non-NK inhibitory 13X3.6 antibodies is substan- tially higher than that of the NK inhibitory 13.1113.3

antibodies. This is shown by the differences (Table 1) in their relative rates of dissociation from the cell surface. Differences in avidity are largely a reflec- tion of differences in the rate dissociation constants (Newman et al., 1983; Mason and Williams, 1980). Hence inhibition of NK function does not result merely from the binding of high-avidity antibodies to NK cell T-200 structures.

These experiments establish that the basis for the inhibition is the ability of the 13.U13.3 antibodies to react with a distinct and unique epitope on the T-200 structure of the NK cell. We therefore felt it was of importance to determine some additional structural features of this family of glycoproteins. Figure 1 presents the immunoprecipitation profile of the 13.3 and 13.5 antibodies from [3H]-galactose-labeled peripheral blood monomrclear cells (left) or the T leukemic cell line CEM (right). This experiment was run under reducing conditions, but non-reducing experiments indicate that the individual bands on polyacrylamide gels are not disulfide-linked to one another. In Fig. 1 we see the characteristic T-cell profile of T-200 on the CEM line, with only the two lower bands in evidence. Resting T-cells, however, exhibit all four bands (see later).

The mobility of the T-200 profile is however slowed as a consequence of reduction by 2ME. Figure 2 shows the reducing and non-reducing

13.3 13.5 13.3 13.5

Fig. 1. Radioimmunoprecipitations with 3H-labeled detergent extracts of PBL (left) or the T leukemic cell line CEM (right). Lysates were treated with antibody coupled to Sepharose. The 6% acrylamide gel was run under reducing

conditions, with myosin as the 200K marker.

profiles of lz51-labeled peripheral blood mono-

nuclear cells in adjacent lanes, and it is clear that each of the four bands is shifted to about the same extent. This result implies the existence of intrachain disulfide bonding, and reinforces the homologous nature of these structures to one another, evident from the peptide mapping studies published by others (Sarmiento et al., 1982; Dunlop et al., 1980). The existence of intrachain disulfide bonds also suggests that these rather large molecules possess a domain structure, as has been shown for immunoglobulin and MHC gene products.

To gain a better understanding of what relationship exists on the cell membrane between the different members of this group, we performed immunoprecipitations on PBL which were treated, prior to labeling with lz51, with the reducible cross-

linking reagent DSP. The results presented in Fig. 3 with the 13.5 antibody show in lanes 1 and 4 the immunoprecipitates obtained from reduced and non-reduced lysates respectively (not cross-linked). Lanes 2 and 5 show the immunoprecipitates obtained from cross-linked and reduced or non- reduced lysates respectively. As can be seen in lane 5, cross-linking has prevented the immunoprecipitate from entering the 6% acrylamide gel. Note that each of the four bands is removed. On reduction of the cross-linked immunoprecipitate, each of the four bands reappears (lane 2), and without any additional labeled proteins. This result tells us that no members of this family exist in the membrane at a distance from some other member greater than about 12 A. What we cannot answer from these experiments is whether all four members are in proximity to one another (assuming all four are represented on a single cell), or whether there are preferential associations between certain members of this group. Non-denaturing gels may give us some insight into this matter. Regardless of that, evidence for some degree of association is evident. It is also of some interest that no additional membrane structures could be shown to associate with T-200 within the range of the cross-linking agent. This makes less likely the possibility that blockade of NK cell activity by anti-T-200 reagents is via interaction with a close neighbor of these high mol. wt glycoproteins, rather than with T-200 itself.

As stated earlier, different mol. wt members of

this family tend to associate with distinct cell types. In particular, the T-cell and macrophage forms of T- 200 have been shown to be quite distinct on murine cell lines (Tung et al., 1981). We sought, therefore, to lend some weight to the argument, pro or con, as to whether NK cells share a T- or a monocytic lineage by examination of the T-200 profile of human NK cells. To examine this question it was necessary to obtain a greatly enriched NK population from peripheral blood mononuclear cells. This we did by Percoll density gradient fractionation of the nylon-wool non-adherent (NWNAD) frac-

1116 WALTER NEWMAN et al

R N N N N R

Fig. 2. Peripheral blood mononuclear cells were surface-labeled with 12sI as previously described (Newman ef al., 1983) and immunoprecipitates with the 13.5 antibody coupled to Sepharose were obtained. Aliquots of these were reduced (R) or left non-reduced (N) and run on a 6% acrylamide gel.

M is the mol. wt marker position for myosin.

tion. The light-density fraction contains the large granular lymphocyte population (LGL), a mor- phological correlate of NK activity. In the high- density fraction are the resting T-cells. Our LGL preparation possessed an NK activity of 33.3 lytic units (LU)/106 cells. One lytic unit is the number of effector cells required to achieve 30% lysis of K562 tumor targets. The starting population of NWNAD cells possessed 2.9 LU/106 cells, and the high- density T-cell fraction 0.7 LU1106 cells. The propor- tions of LGL in the different populations are 8.1% in the starting NWNAD, 4.1% in the high-density (T-cell) and 46.9% in the low-density (LGL) frac- tions. Each population was labeled in its cell surface galactose residues with 3H. A portion of each labeled population was then treated with a low dose of trypsin (5 Kg/ml) prior to detergent lysis. The trypsin treatment gives a unique fragmentation pattern for T-200, depending on the cell type (Fast et

al., 1983). The results, shown in Fig. 4, include T-200

derived from a plastic adherent (mostly monocyte) population of cells. NWNAD, T- and NK cells all show the same four-band pattern, whereas monocytes show only the two bands at 205 and 220K. The trypsin fragmentation profile confirms the similarity of NK T-200 with T-cells and its dissimilarity with monocyte T-200, the latter being resistant to trypsin

at this dose. It is not clear whether this difference in trypsin sensitivity is due to protein structural differences or to differences in the disposition of T-200 in the membrane of T- or NK cells and monocytes.

We next set out to define in more precise terms what stage of the NK-target lytic cycle was affected, with the aim of defining more precisely the role of T- 200 in NK function (Targan and Newman, 1983). Several investigators have studied the stages of NK and cytotoxic T-cell lytic cycles, and a few simple generalizations are possible. First, the initial contact of killer and target cell is an Mg’+-dependent process, but not one that leads irreversibly to delivery of the lytic signal. Disruption of conjugates prior to the second stage leaves undamaged target cells. This second phase is termed the programming for lysis phase, and is a Ca*+-dependent process. Agents which affect Ca*+ physiology, such as verapamil and trifluoperazine block lysis at this point (Hiserodt et al., 1982b; Quan et al., 1982). In this phase, the killer cell, having recognized the target, engages its machinery to deliver the lytic signal. This stage, too, is somewhat reversible in that removal of Ca’+ by chelation, within 20-30 min after its addition, will rescue target cells from lysis. Beyond that period into the third phase, the killer cell independent phase, lysis proceeds beyond the ability of target cells to repair themselves, as

T-200 family of glycoproteins

12 34 5

.

Fig. 3. Peripheral blood mononuclear cells were left untreated (lanes 1 and 4) or treated with the cross- linking reagent DSP (lanes 2,3 and 5), and the immunoprecipitates were run under reducing (lanes 1,2 and 3) or non-reducing conditions (lanes 4 and 5). Cross-linking was performed with 0.5 mM DSP in DMSO-PBS for 30 min at 4”C, followed by several washes. Controls were treated in DMSO-PBS without DSP. Cells were labeled with “‘1 immediately afterwards, as described previously. The control

in lane 3 was done without antibody.

1 2 3 4 5 6 7 8 9 10

1117

Fig. 4. Immunoprecipitations with anti-T-200 antibody 13.4 on lysates from untreated (odd-numbered lanes) or trypsin-treated (5 pg/ml) cells (even-numbered lanes). %I-labeled unfractionated NWNAD cells (lanes 3 and 4), NWNAD cells depleted of NK cells (lanes 5 and 6), NWNAD cells enriched for NK activity (lanes 7 and 8) and adherent cells (lanes 9 and 10). Control immunoprecipitations were performed without antibody on all samples and all were equivalent to the controls shown for lysates

from NWNAD cells (lanes 1 and 2).


demonstrated by experiments wherein target and killer cells are dissociated. Several detailed dissec- tions of the various stages of NK cytolysis have been published (Quan et al., 1982; Hiserodt et al., 1982a).

Our first experiments shown in Table 2 demonstrate quite unambiguously, in three separate experiments, that the NK-blocking antibody 13.3 does not prevent target-killer cell conjugate formation, despite 84-97% inhibition of lysis. Not shown are experiments which demonstrated that the 13.1 antibody does not dissociate preformed conjugates.

The next phase of cytolysis amenable to analysis is the Ca2+-dependent programming for lysis. This

requirement for Ca2+ presumably reflects the existence of a stimulus-secretion coupling mechanism:

the target stimulates the NK cell to secrete soluble lytic moieties within the microenvironment of the target cell (Wright and Bonavida, 1981; Hiserodt et

al., 1983a, b). The precise details of this interaction have yet to be explained (Wayner and Brooks, 1984). This scheme is not inconsistent with a perforin-mediated mechanism of lysis as recently proposed (Podack and Dennert, 1983). We tested the effects of the 13.3 antibody on preformed NK-target conjugates prevented from entry into the programming phase by the withholding of Ca2+. The antibody was found not to disrupt preformed conjugates, and, as long as it was added prior to the addition of Ca*+, even a few minutes before, no lysis of the targets was observed. However, as shown in Fig. 5, there is a very narrow “window of vulnerability” of lysis to inhibition by anti-T-200 monoclonal antibody, which phase we have termed the “trigger” for the cytolytic reaction sequence.

Table 2. 13.1 does not block or dissociate target cell binding“

Percent 51Cr release at Experiment 13.1 3 hrb % TBC’

1 - 61 10.0 (0) _ 61 10.0 (90) + 10 (84) 10.5 (0) + 10 11.0 (90)

_ 12 7.0 (0) + 2 (85) 7.0 (0)

- 39 9.0 (0) - 39 8.5 (90) + 1 (97) 10.0 (0) + 1 8.5 (90)

a Nylon wool non-adherent PBL were mixed with a YIOO dilution of 13.1 and target cells (K562) at E:T ratios of 25:1-4O:l in a regular 4-hr 51Cr release assay, Target binding cells (TBC) were simultaneously determined just after (0) and 90 min (90) after conjugate formation in a standard target binding assay. Before conjugate formation 13.1 was added. In parallel assays, 30-35% of control TBC can lyse bound

b Percent inhibition in parentheses. targets.

c Time after conjugate formation (min) in parentheses,

13 I added

40

30 n zi

H

B 20

6 m

,--o IO

-L-I--_-

-45 0 2 30 60 90 120 I50 I80 210

Duration of Assay Posi -CaC12 irnln)

Fig. 5. Demonstration that 13.1 does not block programming for lysis. PBL and K562 target cells were mixed together at a 35: 1 ratio in EGTAIMg” for 45 min at 37°C to allow conjugated formation. CaCl, (x) or Ab13.1 (0) was added 0, 2, 30, 60, 90, 120, 150, 180 or 210 min after the addition of CaCl,, and the reaction was allowed to continue for a total of 210 min. Supernatants were harvested and tested for 51Cr release. The amount of 5’Cr release occurring at each time when neither EDTA nor 13.1 had been added [kinetics of 51Cr release (0) determined by harvesting supernatants at the indicated time rather than allowing the reaction to go on for 3 hr]. Control “Cr release assay was 61%, and, after addition of 13.1, 10%. This is one of three experiments with similar results.

Hence antibody added simultaneously with CazC still results in inhibition, but if Ca2+ is added as little as 2 min prior to the addition of antibody, no inhibition of lysis is observed. Hence Ca2+ triggers a series of events which, once set in motion, preclude inhibition by the anti-T-200 antibody. Controls presented in Fig. 5 show that simply chelating the Ca2+ in the medium by addition of EDTA also inhibits lysis, but the kinetics are very different from

those seen with addition of antibody. Hence addition of EDTA as late as 90 min after the addition of Ca2+ still gave partial inhibition of lysis, while addition of antibody just 2 min post Ca2+ gave no inhibition of lysis. This result, plus other experiments (not shown) make it unlikely that the 13.3 antibody is competing in any direct way with the binding of Ca*+ to the NK cell, though the antibody does block subsequent Ca2+-mediated effects.

DISCUSSION

T-200 is a major glycoprotein complex whose function(s) on hematopoietic cells has been unex- plained since its first description almost 10 years ago. Based upon data presented here, plus the work of several laboratories, we now possess sufficient structural and physiological information to make some guesses about the role of T-200 in these different cell populations, and to suggest experiments for the future.


To recapitulate, T-200 is a phosphorylated trans- membrane complex of four or five members which are likely the products of distinct but closely related genes, as demonstrated by chymotryptic peptide maps (Tung et al., 1984) and biosynthetic labeling experiments (Watson et al., 1981). They also possess distinctive glycosylation profiles, but carbohydrate alone does not account for all the mol. wt variation. The various members are selectively expressed on hematopoietic cells of distinct lineages, suggesting that they perform distinct but highly related func- tions for the different cell types. Each member of the T-200 complex possesses internal disulfide bonds, which strongly suggests the presence of functionally distinct domains. These glycoproteins are firmly anchored in the plasma membrane, and have a large intracytoplasmic C-terminal region. This, together with the proximity of some if not all of these members to one another, as shown by cross- linking studies, suggest that the T-200 complex is ideally situated for the transmission of signals from the cell exterior to the cytoplasm.

It is of great interest, therefore, that, through an unrelated series of investigations, we developed a monoclonal antibody which regulates such signaling for NK cells interacting with target cells, and that these antibodies are directed to a unique epitope on the T-200 structure, called A. One other exciting feature of these NK-blocking antibodies, presented elsewhere (Newman, 1982), is the specificity of their inhibition, which suggests that the 13.1 and 13.3 antibodies are reacting with an NK cell receptor. Namely, these antibodies block the ability of NK cells to lyse myeloid and erythroid type tumor targets, with no effect on the ability to lyse the standard NK-sensitive T-lymphoma targets. This result has been extended to clones of NK cells which lyse both target types (Pawalec et al., submitted for publication), suggesting that individual NK cells have distinct mechanisms (receptors) by which they recognize different classes of targets. A detailed investigation of the mechanism by which the 13.1 and 13.3 antibodies block NK cell activity has resulted in the definition of a new stage of the NK lytic cycle termed “triggering.” This stage occurs subsequent to the conjugation of effector and target, but prior to the Ca2+-dependent programming for lysis events. Attempts to overcome the blocking effect of the antibodies by addition of the Ca ionophore A23187 has not met with any success, though the drug is quite toxic for NK cells.

A major concern with the postulate that T-200 acts as an NK receptor is the fact that these structures, and the epitope A defined by the blocking antibodies, is widely distributed. It may be incorrect, however, to think of NK cell receptors in the same terms we do for T- and B-cells, whose receptors are allelically excluded. Investigators have been much less successful in demonstrating segregation of NK cell specificity at the clonal level than

has been possible for T- or B-cells, though finding such segregation assumes there is more than one target structure. Generally, NK clones lyse a wide variety of susceptible tumor targets (Dennert et al., 1981; Nabel et al., 1981; Brooks et al., 1982): clones with somewhat more restricted specificities have been described (Minato and Kano, 1984). In other words, NK cell receptors may not be allelically excluded, and we should not rule out that a particular domain of a more widely distributed molecule, perhaps a large and complex one like T- 200, could have a receptor-like function. The “trigger” stage of the lytic cycle, wherein T-200 seems to function, is not inconsistent with this notion.

The Ca2+-staging experiments presented here give additional support to the notion that T-200 performs a receptor function for NK cells. The blocking antibodies have been shown to exert their effect just prior to the Ca2+-dependent stimulus- secretion events. By analogy with other systems, such as mast cell histamine release on binding of IgE antibody-antigen complex, the release of catechol- amine from adrenal medulla upon binding of acetyl- choline, or platelet release of Shydroxytryptamine on binding of collagen, the specific interaction of ligand with receptor immediately precedes the Ca’+-dependent secretory events. Hence the position in the sequence of events wherein antibodies to the A epitope of T-200 exert their effect is entirely consistent with a receptor role for these glycoproteins.

We have also been able to support arguments for the T-cell nature of NK cells with the immunochemical experiments on T-200. Highly enriched NK cells express a profile of these high mol. wt glycoproteins which much more closely resembles T- cells than monocytes, both in size distribution and trypsin sensitivity. This result is in keeping with other reports concerning the expression of T-cell differentiation antigens on NK cells (Kaplan and Callewaert 1978; Fast et al., 1981), and of the close relationship between T- and NK cells demonstrated by experiments with cloned cytotoxic T- and NK cells (Brooks, 1983).

It is interesting to note in the recent study by Yakura et al. (1983) that the Ly 5 structure on B- lymphocytes has been implicated in regulating or participating in the transmission of soluble signals from macrophage to B-cells. It will be most interesting to see if the effects of anti-Ly 5 in this system can be narrowed to some phase of B-cell activation or secretion relative to the requirement for Ca*+. As of yet we have no clues as to the roles which T-200 plays in the function of other hematopoietic cell types, but it would seem from the aforementioned that a reasonable place to look would be proximate to whatever Ca2+-dependent secretory processes these cells can perform. We can say, however that the anti-T-200 reagents 13.1 and 13.3 do not affect lysis by other kinds of cytolytic effecters, including


cytotoxic T-lymphocytes, antibody-dependent cyto- Hiserodt S. C.. Britvan L. J. and Targan S. R. (1982a) lytic effecters, lectin-dependent cytolytic effecters, Characterization of the cytolytic reaction mechanism of

and monocytes (Newman et al., 1983). Perhaps the human natural killer (NK) lymphocyte: resolution

monoclonal antibodies developed to other domains into binding, programming and killer cell independent

of the T-200 structure(s) will aid in defining the steps. J. Immun. 129, 1782-1787.

Hiserodt J. C., Britvan L. J. and Targan S. R. (19828) function of these high mol. wt glycoproteins on

teins will depend upon more detailed structural

other cell types. In the future, therefore, further elucidation of the

role of this complex family of high mol. wt glycopro- Hiserodt J. C., Britvan L. J. and Targan S. R. (1983a)

Differential effects of various pharmacologic agents on the cytolytic reaction mechanism of the human natural killer lymphocytes: further resolution of programming for lysis and KCIL into discrete steps. J. Zmmun. 129, 22662270.

information, particularly amino acid sequence data for the individual members of the group. Such information will help to relate this family to those cell surface molecules whose amino acid sequence is known, and will serve as a basis for cloning the T- 200 genes. It is likely that, in understanding their genetic organization and the control of their expression, we shall proceed most rapidly with the fullest explanation of the function T-200 subserves.

edge the excellent technical assistance of MS-Geraldine Shu, MS Adriene Niederlehner and Mr Kevin Draves. and

AcknowledRemenrs-We would like to gratefully acknowl-

Studies on the mechanism of the human natural kille; cell lethal hit: evidence for transfer of protease-sensitive structures requisite for target cell lysis. J. Zmmun. 131, 2710-2713.

Hiserodt J. C., Britvan L. J. and Targan S. R. (1983b) Studies on the mechanism of the human natural killer cell lethal hit: analysis of the mechanism of protease inhibition of the lethal hit. J. Zmmun. 131, 2705-2709.

Hoshino T., Koren H. S. and Uchida A. (Eds) (1984) Natural Killer Activity and Its Regulation. Excerpta Medica, Amsterdam.

Cancer Inst. 60, 961-964.. Kasai M.. Leclerc J. C.. Shen F.-W. and Cantor H. (1979)

Kaplan J. and Callewaert D. M. (1978) Expression of human T cell antigens by natural killer cells. J. natn.

the expert secretarial assistance of MS Nancy Lawery. This work was supported by grants AI-16496, AI-15332 and AI- 15393 from the National Institutes of Health, by a Veterans Clinical Investigator Award, a National Science Foundation Award PCM8008749, and by the Ortho Phar- maceutical Corporation. In particular, Walter Newman gratefully acknowledges the years of encouragement and support from Elvin Kabat as a student within and outside his laboratory.

Identification of Ly-5 on the surface of ‘natural killer’ cells in normal and athymic inbred mouse strains. Immunogenetics 8, 153-159.

Komuro K., Itakura K., Boyse E. A. and John M. (1975) Ly-5: a new T lymphocyte antigen system. fmmuno- genetics 1, 452-456.

Lutz H. U., Lomant A. J., McMillan P. and Wehrli E. (1977) Rearrangement of integral membrane compon- ents during in vitro aging of sheep erythrocyte mem- branes. J. Cell Biol. 14, 389-398.

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Immunobiological and immunochemical aspects of the T-200 family of glycoproteins