Lens culinaris lectin is a T-cell mitogen: Binding inhibition by concanavalin A and phytohemagglutinin-P

CELLULAR

IMMUNOLOGY

36, 65-74 (1978)

fens culinaris Pectin is u T-Cell Mitogen: Binding Inhibition by Concanavalin A and Phytohemagglutinin-P KEIKO

OZATO,~ DELORES SOMERVILLE,

AND JAMES D. EBERT~

Carnegie Institution. of Washington, Department of Embryology, 115 West University Parkzaay, Baltimore, Maryland 21210 Received August 24,1977 Binding and mitogenicity of a lectin from Lens cz&aris (LcH) were studied in mouse lymphocytes. Both continuous and pulse treatment of lymphocytes with LcH induced a mitogenic response selectively in T cells. LcH and Con A, which have similar binding specificities, exhibited binding inhibition both in unfixed cells and glutarafdehype-fixed cells, with native Con A and succinyl Con A and at 37°C as well as 0°C. On the other hand, reciprocal binding inhibition by a third T-cell mitogen, phytohemagglutinin-P (PHA-P), was found only in unfixed cells at 37°C and with native Con A, indicating that the inhibition is a secondary effect as opposed to direct competition for receptors. The inhibition of mitogenic responses to LcH and PHA-P by pretreatment of cells with Con A was studied in relation to the two different types of binding inhibition, Only the type of binding inhibition caused by a secondary effect correlated with interference with the mitogenic response.

INTRODUCTION Lentil l&tin obtained from Lens cuZ&ris (LcH) is widely used in affinity chromatography for isolating membrane glycoproteins (1) including I-IL-A antigens from human lymphocytes (2) or sialoglycoprotein and Component a from erythrocytes (3). Similar to concanavalin A (Con A) (4) the binding of LcH to cells can be inhibited specifically by a-methyl-D-mannopyranoside, D-nlannOSe, and a-methyl-D-glucopyranoside (5). This lectin has two subunits of molecular weight 24,500 (6), similar to that of the lectin from Pisun% sntivu~ (7, 8). In addition, lentil lectin is known to induce a mitogenic response in human and guinea pig lymphocytes (9, 10). However, it is not known whether LcH induces a mitogenic response selectively in either T or B cells, or in both. Pea lectin and Con A are known to activate T cells selectively (11, 12). In this paper we report that LcH is also a T-cell mitogen as tested in mouse lymphocytes. Based on this information, we studied the binding of ‘251-labeled LcH to thymocytes in detail. Our study of reciprocal inhibition in binding between Con A and LcH reveals that at least some of the lymphocyte receptors for LcH are the same as those for Con A. Binding inhibition was also studied with PHA-P, a T-cell 1 Present address: Johns Hopkins University, School of Medicine, and O’Neill Laboratories, Good Samaritan Hospital, Baltimore, Maryland 21239. 2 Present address : Marine Biological Laboratory, Woods Hole, Massachusetts 02543. 65 0008~8749/78/0361-0065$02.00/O Copyright@ 1978by AcademicPress,Inc. in any form reserved

All rights of reproduction

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mitogen with a different sugar binding specificity (N-acetyl-D-galactosamine) (13). We show that the binding of T-cell mitogens of different sugar specificities to thymocytes can be inhibited reciprocally, not by binding competition for the same receptors but through a secondary effect of Iectin binding such as receptor redistribution, endocytosis, or altered metabolic activity. MATERIALS

AND

METHODS

Mice and cells. Male CBA/J mice, 4 to 8 weeks old, were purchased from Jackson Laboratory (Bar Harbor, Me.). Hydrocortisone sodium succinate (4 mg) was injected 2 days before sacrifice to obtain cortisone-resistant thymocytes (CRT). Thymus cell suspensions were prepared in ice-cold RPMI- 1640 (GIBCO, Grand Island, N.Y.) filtered through silk mesh, washed, and resuspended in an appropriate solution. These cell suspensions were 98y0 viable as detected by the trypan blue dye exclusion test. Athymic mice (nu/nu) were kindly provided by Dr. W. H. Adler (Gerontology Research Center, NICHD, PHS, Baltimore City Hospitals). Lectins. Native Con A (N-Con A) and leucoagglutinin (LA), purified agglutinin from PHA-P (14, 15), were obtained from Pharmacia (Piscataway, N.J.). PHAP was purchased from Difco (Detroit, Mich.). LcH prepared from lentils according to Howard et al. (6) with a slight modification (16) was a generous gift from Dr. J. Cebra. Succinyl Con A (S-Con A) was prepared according to Gunther et al. (17). The lectins were dissolved in appropriate solutions immediately before use. Anti-thy 1.2. Anti-thy 1.2 was prepared by injecting CBA thymocytes into AKR mice according to Reif and Allen (18). To eliminate thy 1.2-positive cells, 1 X 10’ cells were incubated in 1 ml of the antiserum (diluted 1: 40 in medium) in the presence of absorbed rabbit complement (final dilution 1: 15) at 37°C for 45 min. 1251-Labeled lectins. Iodination of lectins was performed by the chloramine-T method (19). lz51-labeled Con A and 1251-labeled LcH were purified by affinity chromatography on Sephadex G-100 and eluted with 0.3 M a-methyl-D-mannopyranoside ((Y-MM) (20). They were further purified by gel filtration on Bio-Gel P-60 or P-100. The lz51-labeled PHA-P was purified on Bio-Gel P-60 equilibrated with 0.05 M Tris, pH 7.2, followed by extensive dialysis. The labeled materials were stored at -20°C in 0.05 M Tris-HCI, pH 7.2, in the presence of 1 mM Ca2+ and 1 mM Mg ZZ+ . A detailed description of lectin iodination is presented elsewhere.3 The labeled lectins were electrophoretically indistinguishable from the unlabeled materials. Specificity of binding of lz51-labeled lectins was tested as follows. Thymocytes were incubated with lz51-labeled Con A (2 pg/ml) for 30 min at 0°C then the cells were further incubated with 0.2 M a-MM at 37°C for 30 min, followed by two washings. Approximately 95% of bound lz51-labeled lectins were eluted. The binding of 1251-labeled PHA-P (5 pg/mI) at 0°C was inhibited by more than 90% when thymocytes were preincubated with unlabeled PHA-P at 500 pg/ml for 30 min. Assay for mitogenic response. Single cell suspensions of CRT from CBA/J or spleen cells from athymic mice prepared in the medium RPM1 * 1640 supplemented 3 K. Ozato and J. D. Ebert (1977). The retention of cell bound mitogens during blastogenic transformation. Submitted for nublication.

T-CELL

MITOGEN

67

LCH

with appropriate concentrations of fetal calf serum (FCS) (GIBCO) and gentamycin at 50 pg/ml were cultured at a cell concentration of 2 X 105/0.5 ml in plastic culture tubes (Falcon Plastics ; 10 x 75 mm) in the presence or absence of lectins, in humidified gas composed of 83% Nz, 10% COZ, and 7% 02 for 72. hr. After various periods of incubation, cells were labeled with [3H]TdR at 1 pCi/ml (sp. act., 14 Ci/mmol; Schwartz/Mann) for 18 to 20 hr. Incorporation of [aH]TdR into 50/o trichloroacetic acid-insoluble material was measured in a liquid scintillation spectrometer. Lymphoblasts were determined by staining the harvested cells with Giemsa ; enlarged cells (of at least twice the diameter of small lymphocytes) with dinstinct cytoplasmic space were counted as lymphoblasts. Binding of 1251-labeled Zectins to thyynocytes. Four million thymocytes or CRT were incubated in the presence of 1-5 pg/ml of lz51-labeled lectins diluted with unlabeled lectins to a convenient specific activity in Gey’s balanced salt solution (GBSS)depleted of glucose. To prevent adsorption of lz51-labeled lectins to glass, tubes were pretreated with 0.1% bovine serum albumin. For the study of binding inhibition 4 x lo6 cells were preincubated with unlabeled lectins at various concentrations in 0.3 ml of GBSS without glucose for 20 min at 0 or 37°C. Then 0.1 ml of a solution of lZ51-labeled lectins was added, and the cells were incubated for another 40 min unless otherwise stated. Duplicate tubes were prepared for each measurement. To obtain fixed cells, thymocytes were fixed with 2.5yo glutaraldehyde in RPMI. 1640 at room temperature for 15 min and washed three times with a large volume of RPMI. 1640. Since some lectins facilitated the adsorption of other 1251-labeIed lectins to the glass tubes, control tubes without cells were always prepared and carried through the procedure. After incubation cells were washed

CRT

o-0

Pulse (30’ )

-Continuous

\

\

\ ‘\

Nude Spleen 2

‘\ 1

LcH

Concentration(pq/ml

1

FIG. 1. T-cell mitogenic responseinduced by LcH. CRT or nu/nu spleen cells were cultured in the continuous presenceof LcH in medium plus 5% FCS for 72 hr (closed symbol with solid line). Different sets of cells were pretreated (open symbol with dotted line) with LcH for 30 min at 37°C and then indubated in culture medium free of lectin for 72 hr. Each value represents the mean of duplicate measurements of [8H]TdR incorporation after 72 hr of culture.

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three times by centrifugation with ice-cold GBSS without was measured in a gamma spectrometer.

glucose. Radioactivity

RESULTS 1. LcH Induces a iW;togenic Response Selectively in T Lymphocytes CRT or nu/nu spleen cells cultured for 72 hr in medium containing 5% FCS and various concentrations of LcH were tested for their proliferative response as measured by [“HJ TdR incorporation (Fig. 1). Five micrograms per milliliter of LcH stimulated CRT to significant DNA synthesis. More than half of the viable cells were morphologically distinct lymphoblasts with an enlarged cytoplasm, A proliferative response in CRT was induced only over a narrow concentration range, i.e., from 5 to 10 pg/ml. CRT responded even when the cells were exposed only briefly to LcH for 30 min at 37°C followed by subsequent incubation in culture medium without lectin, although higher concentrations (25 to 2.50 pg/ml) of the lectin were required for this activation. The degree of mitogenic response after brief exposure was comparable to continuous exposure (about 80%). The cells showed significant DNA synthesis even when cultured in medium without FCS, but the optimal lectin concentration was shifted to 1 to 2 pg/ml. DNA synthesis by CRT reached a peak after 2 to 3 days of incubation and ceased at 6 days. Normal spleen cells exhibited about the same amount of DNA synthesis as did CRT in the same concentration range, but normal thymocytes responded less vigorously (l/S to l/7 of the CRT response). The nu/nu spIeen ceils did not respond at any concentration of LcH tested, either after pulse treatment or by continuous exposure. Furthermore, the mitogenic response was completely abrogated when CRT or normal thymocytes were treated with anti-thy 1.2 (6) for 45 min in the presence of rabbit complement before stimulation with LcH. 2. Binding

of 1251-Labeled LcH to ThyYlzocytes

LcH at the mitogenic concentraKinetics. We studied the binding of Y-labeled tion of 2 pg/ml to lymphocytes at 37 and at 0°C. A comparison was made between normal thymocytes and CRT which differ in the degree of mitogenic response to the lectin. Figure 2 indicates the kinetics of binding. At 37°C binding increased sharply within 60 min, then the increase slowed but continued until 120 min of incubation. At 0°C the binding appeared to reach a steady state at 60 to 90 min at a Ievel of binding about half that at 37°C. There was virtually no difference in LcH binding between normal thymocytes and CRT at both temperatures. From the specific activity of LcH (Fig. 2) and its known molecular weight of 49,000 (6), it follows that at 60 min about 5.5 x lo5 molecules of LcH bound to a lymphocyte at 37”C, and 3.5 x lo5 at 0°C. In comparison with the binding of 1Z51-labeled Con A, about four times more LcH bound to the cells at the same concentrations in 60 min of incubation at 37°C. However, bound LcH was more readily dissociated from the cells than Con A during subsequent incubation: Half of the bound LcH was released in 90 min, while only 10-13s of bound Con A was eluted in the same time. Reciprocal binding inhibition between Cojt A and LcH. We studied the binding competition between LcH and Con A, As shown in Fig. 3A, 1251-labeIed LcH binding at mitogenic concentrations was inhibited by preincubating the cells with

T-CELL

MITOGEN

M

0

120

30

60 Incubation

69

LCH

(Min)

FIG. 2. Kinetics of ?-labeled LcH binding to thymocytes. Normal thymocytes or CRT were incubated with LcH at 2 pg/ml (sp. act., 2.7 X 10’ cpm/pg) in GBSS without glucose for various periods of time. Each value represents the mean of duplicate measurements.

native or succinyl Con A at 37 or at 0°C. Succinyl Con A at 0°C exhihited only a slight degree of inhibition which was nevertheless detectable. Inhibition in glutaraldehyde-fixed cells both at 37 and 0°C occurred to the same extent as inhibition at 0°C in viable cells. Binding inhibition of l”jI-labeled Con A by LcH (Fig. 3B j was observed similarly at both 37 and O”C, regardless of whether or not the cells were fixed. The hinding inhibition was not due to the inactivation of one lectin by the other, since a similar degree of binding inhibition was detected when the unbound lectin of the first treattnent at a concentration of 25 &ml was washed -. away before application of the labeled lectin.

-a----

.._-__-_

* .------, O°C o*c

*4 WC

$-a S-ConA

-

N-ConA

37-C N-Con A 5

I

0

A

404 25

0

6, 10

LcH

I

I

20

30

Concentration

40

40

‘+k-

(,ug/ml )

FIG. 3. Reciprocal binding inhibition between Con A and LcH: (A) inhibition of ‘“I-labeled LcH binding by unlabeled Con A ; (B) inhibition of mI-labeled Con A binding by unlabeled IxH. Cells were preincubated with various concentrations of Con A, native (N-1 Con A, or succinyl (S-) Con A (A) or LcH (B) for 20 min at 37 or 0°C and were further labeled with 2 pg/ml of 1’61-labeled LcH (A) (sp. act., 2.5 X 10’ cpm/&g) or ‘“I-labeled Con A (B) (sp. act., 4.6 X IO’ cpm/pg) for 40 min at 37°C. The values represent the means of duplicates.

70

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UnfIxed

37OC

A UnfIxed n

AND

EBERT

_

0-C

Fixed

37OC

\

_

A Unfixed n

O’C

Fixed 37°C

. l

4+-•

a

s ” f

50‘z51-CcnA 40*. 0

: I pq/ml

4 c

A IO LA

20 30 Concentration

40

50 (Ag/ml)

”

LB

0

200

10

5

20

PHA.P

I_j

40

Concentration(pg/ml)

FIG. 4. Inhibition of ?-labeled Con A binding (A) or “I-labeled LcH binding (B) by unlabeled LA or PHA-P. Cells preincubated with LA (A) or PHA-P (B) were further labeled with 12SI-labeled Con A (2 pg/ml; sp. act., 4.6 X lo4 cpm/pg) or Y-labeled LcH (2 ,ug/ml ; sp. act., 2.5 X lo4 cpm/pg). The values represent means of duplicates.

Reciproca’l binding inhibition between Con A or LcH and PH,4-P. Next, binding inhibition was studied with PHA-P, another T-ceI1 mitogen with a monosaccharide specificity (N-acetylgalactosamine) different from that of Con A and LcH. In some experiments we used LA which is a more highly purified form of PHA-P (14, 15). Figures 4A and I3 show the inhibition of the binding of 1251-labeled Con A or 1251labeled LcH when cells were pretreated with unlabeled PHA-P or LA. The binding of both 1251-labeled lectins was significantly inhibited at 37°C with viable cells. However, no inhibition was found either when cells were fixed with glutaraldehyde and tested at 37°C or when unfixed cells were tested at O”C, in contrast to the inhibi-

OT N-Con A --$/-A --_ --% S-ConA -----------O-----*----a 3PC S-ConA

.L

37OC N-ConA

f-

0

I

25 Lecttn

1

I

50 75 Concentration

I

100 @g/ml

A‘

I

”

200 1

FIG. 5. Inhibition of Y-labeled PHA-P binding by pretreatment with unlabeled Con A or unlabeled LcH. Cells preincubated with N-Con A or S-Con A at 0 or 37°C were further labeled with ‘*SI-labeled PHA-P (5 pg/ml ; sp. act., 5.3 X 10’ cpm/pg). The values represent means of duplicates.

T-CELL

MITOGEN

71

LcH

ConA PrCtrcatment N-CanA S -ConA ---Mitogen

Stimulation

Continuous LCH 0 LA A

ConA

Concentration

Pulse . .

(pg/mi)

FIG. 6. Suppression of mitogenic response to LcH or LA by pretreatment of the cells with N-Con A. CRT were preincubated with various concentrations of N-Con A or S-Con A in RPMI.1640-k 10% FCS for 40 min at 37°C. After removal of unbound Con A by washing, the cells were stimulated with a pulse treatment of LcH (25 fig/ml) or LA (50 pg/ml) for 30 min and incubated in culture medium for 72 hr. Alternately, the cells were stimulated by continuous incubation in LcH (2.5 pg/ml) or LA (10 pg/ml). 13H]TdR incorporation after 72 hr is shown. The values represent means of duplicates.

tion between Con A and LcH. The reciprocal experiment, that is, binding of lz51labeled PHA-P at 5 pg/ml after preincubation of the cells with unlabeled Con A or LcH, is shown in Fig. 5. In the absence of a competing lectin about 14 or 8 ng of PHA-P were bound to 1 X lo6 cells at 37 or O”C, respectively. The inhibition was again temperature dependent ; at 0” C, LcH or Con A did not inhibit the subsequent binding of PHA-P. The inhibition was dependent further on the valence of Con A, in that S-Con A at 37°C was inhibitory only by 10%. Thus the pattern of the binding inhibition between Con A or LcH and PHA differs from that between Con A and LcH. Inhibition of the ktogenic response to LcH or LA by N-Con A pretreatment. We have found previously that the mitogenic response to Con A was suppressed by PHA-P pretreatment (21). In order to delineate further the relationship between binding inhibition and suppression of mitogenesis, CRT were pretreated with NCon A or S-Con A for 30 min prior to subsequent mitogenic stimulation by LA or LcH. In our culture conditions CRT alone do not respond to a brief treatment with Con A at any concentrations (22, 23). However, CRT can respond to a brief Con A treatment in cell-Mediated ktogenic response, as described elsewhere (23, 24). As shown in Fig. 6, N-Con A pretreatment reduced the mitogenic response to a subsequent stimulus by LcH or by LA. Complete abolishment of mitogenic response to either LA or LcH was found after pretreatment at 25 pg/ml of N-Con A or at higher concentrations, regardless whether the subsequent mitogen treatment was brief or continuous. In contrast, S-Con A pretreatment showed no sup-

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pression of the response to LA or LcH. Even at high concentrations such as 100 pg/ml of S-Con A, neither inhibition nor enhancement was detected in the response to either mitogen. The reversibility of suppression was shown in the following way: When CRT were treated with 0.2 M a-MM for 20 min at 37°C immediateIy after N-Con A pretreatment and before exposure to LcH or LA, the mitogenic response to LcH or LA was restored to a normal level. This suggests that the suppressive effect is due to Con A bound to the cells rather than to any immediate cytotoxicity of this Iectin. DISCUSSION In the first part of the present paper we report that LcH is a T-cell mitogen. We base this conclusion on the lack of mitogenic responsiveness of splenic lymphocytes from athymic mice and the susceptibility of the response to anti-thy 1.2. T cells are activated even by brief exposure to the lectin. Furthermore, like other T-cell mitogens Con A and LA, LcH induces a cell-wtediated mitogenic re.+onse in CRT stimulated by syngeneic lymphocytes as described elsewhere (23, 24). Because the binding of 1251-labeled Con A and 1251-labeled LcH were reciprocally inhibited by the other unlabeled lectin LcH must share, at least in part, common receptors with Con A. The characteristics of LcH binding and Con A binding are similar in that they bind to CRT and normal thymocytes to a similar extent and with similar kinetics, at least at mitogenic concentrations. Furthermore, our previous autoradiographic analysis 3 indicated that in the thymocyte population individual cells bind to either 1251-labeled LcH or 1z51-labeled Con A in a reasonably homogeneous manner, as tested at mitogenic concentrations. But, in terms of stability, the LcH binding is more heterogeneous than that of Con A. Reciprocal binding inhibition was studied not only between Con A and LcH, which have the same monosaccharide binding specificity (4, 5, 9), but also between Con A or LcH and PHA-P that has a different monosaccharide specificity (13). Our results indicate that binding of Con A or LcH is significantly inhibited by PHA-P and vice versa (21). However, binding inhibition between Con A and LcH differs in its basic mechanism from the inhibition between Con A or LcH and PHA-P in the following ways. (i) Con A-LcH interference occurs at 0 and 37”C, whereas inhibition between Con A (or LcH) and PHA-P occurs only at 37°C. (ii) The first type of inhibition is found whether or not the cehs were fixed, but the second type requires intact cells. (iii) Interference between Con A and PHA-P is dependent on Con A valence. S-Con A inhibits LcH binding profoundly, but PHA-P binding weakly. These results show that there are two types of reciprocal binding inhibition : The first is a simple competition for common receptors, while the second is due to membrane alteration induced secondarily by the binding of an unrelated lectin. There are at least three conceivable mechanisms for the second type of inhibition: (i) Receptor redistribution such as clustering or cap formation (25, 26) that occurs after lectin binding and is temperature dependent could sequester receptors for the unrelated lectin. It is known that the binding of a lectin to lymphocytes controls the mobility of receptors for other ligands (27, 28). Binding of such ligands to their receptors could also be affected by membrane redistribution. (ii) The mechanism may involve endocytosis (25, 26) that follows ligand binding at 37°C. The internalization of the first Iectin could induce membrane remodeling (29, 30) on a large scale; as a result, existing membrane components could become

T-CELL

MITOGEN

LCli

73

inaccesible or part of them may be concomitantly internalized. (iii) Binding inhibition might be regulated by intracellular events. There are numerous changes in intracellular metabolic activity immediately following ligand binding (3133). Such events could affect membrane organization, synthesis, or turnover. Irrespective of mechanism, the second type of binding inhibition apparently results in a more profound internal effect in that it seems to lead the responsiveness to subsequent mito genie signaling (34) (see Fig. 6). Using three mitogenic lectins, this work provides evidence for characteristic binding inhibition distinct from receptor competition induced secondarily by membrane alteration. This type of inhibition may be the mechanism in certain types of immune unresponsiveness as suggested by Moller (35). ACKNOWLEDGMENTS We gratefully acknowledge Dr. J. Cebra for providing us purified LcH and for his helpful comments, and Dr. I. B. Dawid for his critical reading of the manuscript. We also thank MS. B. Smith for her valuable technical assistance.

REFERENCES 1. Hayman, M. J., and Crumpton, M. J., Biochem. Biophys. Rex COVWWL 47, 923, 1972. 2. Bridgen, J., Snary, D., Crumpton, M. J., Branstable, C., Goodfellow, P., and Bodmer, W. F., Nature (London) 261, 200, 1976. 3. Findlay, J. B. C., J. Biol. Chem. 249, 4398, 1974. 4. Powell, A. E., and Leon, M. A., Exp. Cell Res. 62, 315, 1970. 5. Stein, M. D., Howard, I. K., and Sage, H. J., Arch. B&hem. Biophys. 146, 353, 1971. 6. Howard, I. K., Sage, H. J., Stein, M. D., Young, N. M., Leon, M. A., and Dyckes, D. F., /. Biol.

Chew

246, 1590, 1971.

7. Trowbridge, I. S., J. Biol. Chew 249, 6004, 1974. 8. Trowbridge, I. S., Proc. Nat. Acad. Sci. USA 70, 3650, 1973. 9. Young, N. M., Leon, M. A., Takahashi, T., Howard, I. K., and Sage, H. J., J. Biol. Cltem. 246, 1596, 1971. 10. Ahmann, G. B., and Sage, H., J. Cell. Inmmnol. 10, 183, 1974. 11. Trowbridge, I. S., Ralph, P., and Bevan, M. J., Proc. Nat. Acad. Sci. USA 72, 157, 1975. 12. Andersson, J., Moller, G., and Sjoberg, O., Cell. Imm~nol. 4, 381, 1972. 13. Borberg, J. H., Woodruff, J., Hirschhorn, R., Gesner, B., Miescher, P., and Silber, R., Science 154, 1019, 1966. 14. Weber, T., Scand. J. Clin. Lab. Invest. Suppl. 111, 33, 1969. 1.5. Skoog, V. T., Weber, T. H., and Richter, W., Exp. Cell Res. 85, 339, 1974. 16. Ticha, M., Etlicher, G., KoStii, J. V., and Kocourek, J., Biochiw Biophys. Acta 221, 282, 1970. 17. Gunther, G. R., Wang, J. L., Yahara, I., Cunningham, B., and Edelman, G. M., Proc. Nat. Acad. Sci. USA 70, 1012, 1973. 18. Reif, A. E., and Allen, J. M., J. Exp. Med. 120, 413, 1964. 19. Greenwood, F. C., Hunter, W. M., and Glover, J. S., Biochenz. J. 89, 114, 1963. 20. Cuatrecasas, P., Biochemistry 12, 1312, 1973. 21. Ozato, K., Ebert, J. D., and Adler, W. H., J. Imnrunol. 115, 339, 1975. 22. Lindahl-Kiessling, K., Exp. Cell Res, 70, 17, 1972. 23. Ozato, K., and Ebert, J. D., I. Exp. Med. 143, 1, 1976. 24. Ozato, K., Cebra, J., and Ebert, J. D., J. Exp. Med. 146, 776, 1977. 25. Taylor, R. B., Duffus, W. P. H., Raff, M. C., and de Petris, S., Nature Nezw Biol. 233, 225, 1971. 26. Unanue, E. R., Perkins, W. D., and Karnovsky, M. J., J. Exp. Med. 136, 885, 1972.

27. Yahara, I., and Edelman, G. M., Proc. Nat. Acad. Sci. USA 72, 1579, 1975.

74

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SOMERVILLE,

AND

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28. Hellstriim, U., Diller, M. L., Hammestrom, S., and Perlman, P., Stand. 1. Immum~Z. 5, 45, 1976. 29. Unanue, E. R., Ault, K. A., and Karnovsky, M. J., J. Exp. Med. 139, 295, 1974. 30. Gonatas, N. K., Gonatas, J. O., Stieber, A., Antoine, J. C., and Avrameas, S., I. Cell. E&l. 70, 477, 1976. 31. Schmidt-Ullrich, R., Wallach, D. F. H., and Ferber, E., Biochim. Biophys. Actu 356, 288, 1974. 32. Hadden, J. W., Hadden, E. M., Haddox, M. K., and Goldberg, N. D., Proc. Nat. Acad. Sci. USA 69, 3024, 1972. 33. Painter, R. G., and White, A., Proc. Nat. Acad. Sci. USA 73, 837, 1976. 34. Sidman, C. L., and Unanue, E. R., J. Exf. Med. 144, 882, 1976. 35. Moller, G., Stand. .I. Immunol. 5, 583, 1976.