Formation of heterophile antibodies by human tonsilar lymphocytes

CELLULAR

IMMUNOLOGY

58,246-256

(1981)

Formation of Heterophile Antibodies by Human Tonsilar Lymphocytes’ II. In vitro Stimulation TSUNEATSU

Department

MORI,*

of Microbiology,

with Allogeneic

KYOICHI KANO, JOSEPH AND FELIX MILGROM~

Cells

M. MERRICK,

School of Medicine, State University Buffalo, New York 14214

Received April

of New York,

16. 1980; accepted June 17, 1980

Short-term cultures of human tonsilar lymphocytes (HTL), 5 X lo6 cells/culture, in medium RPM1 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (Xl-300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10’ and 5 X 106/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.

INTRODUCTION Our previous study (1) has shown that short-term cultures of peripheral blood lymphocytes (PBL) from infectious mononucleosis patients formed Paul-Bunnell (P-B) antibodies. It was also shown that human tonsilar lymphocytes (HTL) stimulated in vitro by bovine red blood cells (BRBC), sheep red blood cells (SRBC), or solubilized Hanganutziu-Deicher (H-D) antigen produced heterophile antibod’ Supported by Research Grants AM- 17317 from the National Institute of Arthritis, Metabolism and Digestive Diseases and CA-25852 from the National Cancer Institute. * Tsuneatsu Mori is a recipient of a Henry C. and Bertha H. Buswell Fellowship. ’ Please address all correspondence to Dr. F. Milgrom, Department of Microbiology, 203 Sherman Hall, State University of New York, Buffalo, N.Y. 14214. 246 0008-8749/8 l/040246-1 1$02.00/O Copyright 8 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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241

ies of H-D specificity and that Forssman antibodies were formed by HTL stimulated by SRBC (2). No definite evidence for formation of P-B antibodies was obtained but it appeared likely that some antibodies were directed against antigens of BRBC and/or SRBC other than the three mentioned heterophile antigens. Our observations (3, 4) as well as those of others (5-7) that sera of recipients of skin and kidney allografts contained antibodies combining with xenogeneic erythrocytes suggested that alloimmunization in vivo may also result in formation of “heterophile” antibodies. Pursuing this line of investigation, we found that cultured HTL but not PBL or spleen cells produced plaque-forming cells (PFC) against SRBC and/or BRBC in response to stimulation in vitro by various allogeneic cells. MATERIALS

AND METHODS

Media

Medium RPM1 1640 (Associated Biomedic Systems, Inc., Buffalo, N.Y.) was supplemented with penicillin (100 U/ml), streptomycin (100 pg/ml), fungizone (2.5-5.0 pg/ml) and L-glutamine (1-2 mM). For cultures, this medium was also supplemented with 10% heat-inactivated human AB serum obtained from healthy staff members of this department. Medium 199 (Gibco, Grand Island, N.Y.) was used for PFC assay. HTL Preparation

HTL were prepared from minced tonsils by means of Ficoll-Hypaque gradient separation (8). The exact procedure was described in previous publications (1, 2). The separated mononuclear cells were washed three times with the medium. Stimulators

Tonsilar cells were prepared as described above. PBL were separated from peripheral blood by means of Ficoll-Hypaque gradient centrifugation (8). Splenic mononuclear cells were prepared from spleens removed from patients with lymphomas or leukemias; a procedure similar to that used for separation of HTL was followed. Thymocytes were prepared in a way similar to thymus tissue removed from 2- to 6-year-old children at cardiac surgery. Human AB erythrocytes were obtained from healthy volunteers. Pokeweed mitogen (PWM, Gibco) was used at a final concentration of 10 pg/ml. Lipopolysaccharide (LPS) from E. coli 055:B5 (Difco Laboratories, Detroit, Mich.) was used at a final concentration of 50 pg/ ml. Bactophytohemagglutinin P (PHA, Difco) was used at a final concentration of 10 pg/ml after solubilizing original powder with 5 ml medium. Mitomycin C treatment of lymphocytes was accomplished by incubating lymphocytes at a concentration of 2 X lO’/ml with mitomycin C at a final concentration of 25 pg/ml for 30 min at 37°C. Induction

of PFC in Cell Cultures

Cultures of HTL were established following the previously described procedure (1, 2). They were prepared in 35 X lo-mm plastic petri dishes (Falcon, Oxnard, Calif.) containing 5 X lo6 or 10’ viable HTL in 2.5 ml culture medium with or

248

MORI

ET AL.

without a stimulator. Two days after the initiation of cultures, 0.5 ml of the culture medium was gently added to each culture. Following varying incubation periods in a humidified atmosphere of 5% CO1 in air at 37°C cultures were pelleted and the cells were washed once with medium 199 prior to PFC assay. Collection of Supernatant from Mixed HTL Cultures and Test for Its Enhancing Effects on Allogeneic Stimulation HTL, lo* cells, were cultured together with an equal number of allogeneic HTL or thymocytes in a 30-ml tissue culture flask (Corning Glass Works, Corning, N.Y.) instead of a plastic dish. From these cultures, supernatant fluids were recovered after 24-hr cultivation by centrifugation at 800g for 15 min. To study the effect of the supernatant fluids, the procedure of Rubin et al. (9) was followed; accordingly, supernatant fluids at various concentrations were added to another mixed HTL culture on the second day of culturing. PFC Assay Assays were performed following the previously described procedure (1, 2, 10, 11) with a minor modification. PBL, spleen cells, and thymocytes did not produce any PFC under the present experimental conditions when they were used as responders to mitomycin C-treated allogeneic cells. Therefore, for mixed cultures, PBL, splenocytes, and thymocytes were used without mitomycin C treatment as stimulators of HTL. In contrast, allogeneic HTL were used after treatment with mitomycin C in one-way stimulation experiments. In all these experiments, 2 x lo6 of a cell mixture was incorporated into an agar plate for PFC assay. On the other hand, in two-way stimulation experiments, HTL were untreated with mitomycin C; in these experiments, lo6 of cell mixture were used for PFC assay. Inhibition of PFC Solubilized heterophile antigens were used to determine specificities of antibodies secreted by PFC. P-B and H-D antigens were extracted from BRBC stromata (12) and F antigen was extracted from guinea pig kidney. Acetone powders of guinea pig kidney were suspended in water (1 mg/ml) and vigorously stirred with six parts chloroform:methanol(2: 1) for 2 hr at room temperature. The lower layer was evaporated to dryness and the residue was dissolved in a small volume of chloroform:methanol (2: 1) and the glycolipids were precipitated with 10 vol of acetone. The precipitate was collected by centrifugation, washed several times with acetone, and dried. Finally, the glycolipids were purified as acetylated derivative by chromatography on Florisil as described by Laine et al. (13). The deacetylated purified glycolipid which contained the F antigen was used for subsequent analyses. Human serum albumin (HSA) (Miles Laboratories, Kankakee, Ill.) was used for controls. For inhibition of PFC expression, cultured lymphocytes, target BRBC, and solubilized heterophile antigens or HSA were incorporated into the agar. Thereafter, the PFC assay was followed. The concentration of these antigens was 50.020 mg/ml for the heterophile antigens and HSA.

ANTIBODY

Preparation

FORMATION

BY ALLOGENEIC

of Cells Adherent and Nonadherent

STIMULATION

249

to Plastic

In some experiments, cells adherent to plastic were separated from HTL, according to the slightly modified procedure of Mosier (14). Briefly, 20 ml of HTL suspension at a 2 X lO’/ml concentration in the medium RPM1 1640 containing 5% human AB serum was placed in a 100 x 1j-mm plastic petri dish and incubated without agitation for 1 hr in a COz. incubator. Nonadherent cells were recovered after gentle shaking of the dish and washing once with medium. Adherent cells were then collected by scraping the dish with a rubber policeman. Both cell populations were washed twice with medium. Collection

of Nylon- Wool-Adherent

and Efpuent

Cells

After removal of plastic-adherent cells, 2 X lOa nonadherent cells were passed through the nylon wool column and nylon-wool-effluent cells (T cells) were collected according to the procedure of Julius et al (15). Nylon-wool-adherent cells (B cells) were released from nylon wool with mechanical agitation and collected. Both cell populations were washed twice with the medium and used in culture. Preparation

of E-Rosetting

and Non-E-Rosetting

Cells

E rosettes were formed in HTL suspensionsby using the technique of Bach (16) modified in our previous study (2). The nonrosetting cells (B cells) at the interface and rosetting cells (T cells) at the bottom of a Ficoll-Hypaque gradient were collected separately. Contaminating SRBC in these suspensions were destroyed by the treatment with 0.83% ammonium chloride solution and then cells were washed twice with the medium. RESULTS To find the optimal dose of stimulator cells, a constant number, 5 X 106, of responder HTL were cultured for 6 days with various numbers of allogeneic HTL or PBL. As shown in Fig. 1, the optimal dose of the stimulator HTL was lo’/ culture and of the stimlator PBL 5 X 106/culture. Doses of 5 X lo6 and lO’/culture were then employed for other stimulators: splenocytes, thymocytes, and group AB erythrocytes. Figure 2 shows time kinetics of the appearance of PFC against SRBC after stimulation of HTL with the optimal dose of allogeneic HTL or PBL. A significant number of PFC appeared 3 days after the stimulation by HTL. PFC number reached its peak of 380/culture on the 6th day, and then decreased between the 6th and 10th day. A similar pattern of kinetics was observed in the HTL cultures stimulated by PBL. A total of 55 HTL specimens was examined for formation of PFC against SRBC and BRBC on the sixth day of culturing with allogeneic lymphocytes from various organs. As seen in Table 1, of these HTL, 48 specimens produced a significant number of PFC and the remaining 7 failed to do so. Stimulation by spleen lymphocytes from lymphoma or leukemia patients gave the highest number of PFC against SRBC and stimulation by thymocytes gave the lowest number. The numbers of PFC were approximately equal after stimulation by HTL and by PBL. The

250

MORI

FIG. 1. Stimulation X HTL)

and PBL (HTL

ET AL.

of HTL, 5 X IO6 cells/culture, by various numbers of allogeneic HTL (HTL X PBL). PFC assays were performed with SRBC on the sixth day of culturing.

treatment of stimulator HTL with mitomycin C decreased the number of PFC. In contrast, similar treatments of PBL and spleen lymphocytes did not change the number of PFC significantly and mitomycin C treatment of thymocytes increased the number of PFC. When stimulated HTL cultures were tested against BRBC as target cells, a significant number of PFC was noted, even though it was lower than the number of PFC against SRBC. The majority of control cultures without stimulator cells produced 10 or less PFC/culture and only a few cultures produced 50 PFC. In addition, 10 HTL samples were cultured with group AB erythrocytes or various mitogens such as LPS, PWM, and PHA. No significant numbers of PFC were noted in these cultures. HTL obtained from two patients, PS and GA, were stimulated by PBL of six different individuals. As seen in Table 2, the HTL from PS produced PFC against SRBC but not BRBC after stimulation with any of the PBL specimens. On the other hand, HTL from GA after stimulation with the same five PBL specimens produced, in all instances, higher numbers of PFC against BRBC than against SRBC and in one instance, stimulation with PBL (AS) failed to result in any noticeable PFC against SRBC. Specificities of heterophile antibodies secreted by the PFC of the HTL from PS and GA were studied by inhibition tests using solubilized heterophile antigens (Table 3). Expression of PFC against SRBC produced by HTL of PS was completely inhibited by F antigen at concentrations of 0.34.02 mg/ml, whereas HD antigen failed to inhibit the PFC of PS at these concentrations. In contrast,

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251

STIMULATION

400' " z u) ':

300.

'0 P : 2 5 e " ::

200'

100 .

1

2

3

4

Dmyr

5

after

6

initiation

7 of

6

9

10

cultures

FIG. 2. PFC response of human tonsilar lymphocytes stimulated in vitro by allogeneic lymphocytes. 5 X lo6 cells/culture were stimulated by 10’ mitomycin C-treated allogeneic HTL and 5 X lo6 HTL. PFC assays were performed with SRBC.

expression of PFC against BRBC produced by HTL of GA was completely inhibited by H-D antigen at concentrations of 0.3-0.02 mg/ml but not by F antigen. P-B antigen failed to influence significantly the expression of PFC of both HTL specimens. It appeared, therefore, that the major population of antibodies secreted from TABLE PFC Responses of Human Tonsilar

Lymphocytes

1 Stimulated

by Allogeneic

Lymphocytes

Average No. of PFC/culture Target cells No. of cultures

SRBC (mean * SD)

BRBC (mean _+ SD)

1

277 k 194 137 f 64

84 f 80 53 f 99

5 4

224 f 275 f

spleen* spleen* (MC)

8 3

312 + 248 283 f 126

83 f 70 80 f 26

thymus thymus (MC)

6 2

61 + 14 158 + 67


Stimulator

cells

Lymphocytes from: tonsil tonsil (MC)O peripheral peripheral

blood blood (MC)

13

’ Mitomycin C-treated stimulator cells. b Spleens from lymphoma or leukemia patients.

145 189

126 f 143 170 * 173

252

MORI ET AL. TABLE 2 PFC Responsesof Human Tonsilar Lymphocytes Stimulated by Allogeneic Peripheral Blood Lymphocytes’ PFC/culture stimulated by PBL of: AS

RM

PJ

MC

PL

cw

Responder

Sb

B’

S

B

S

B

S

B

S

B

S

B

PS

195

110

205

<10

165

t10

355


310


320


GA


90

100

515

30

135

115

265

80

225

60

225

a Treated with mitomycin C. b Target cells: SRBC. ’ Target cells: BRBC.

the PFC of PS are of F specificity and those from the PFC of GA are of H-D specificity. Subsequently, the role of E-rosetting cells or T cells in the PFC formation by a mixed HTL culture was studied. As seen in Table 4, removal of E-rosetting cells from both HTL populations resulted in 90% reduction of PFC. In contrast, removal of E-rosetting cells from one population did not affect the PFC formation or it even showed some enhancing effect on PFC formation. These results indicated that T cells were needed in at least one of the HTL populations for the PFC formation. TABLE 3 PFC Responsesof Human Tonsilar Lymphocytes Stimulated by Allogeneic Peripheral Blood Lymphocytes: Inhibition of PFC Expression by Solubilized Heterophile Antigens HTL cultures from Ps

GA

Inhibitors

PFC against SRBC/culture

PFC Against BRBC/culture

H-D antigen’ (mg/ml) 0.3 0.02

315 319


F antigenb 0.3 0.02


255 320

P-B antigen’ 0.3 0.02

320 315

180 230

None

380

330

a H-D: Hanganutziu-Deicher. b F: Forssman. c P-B: Paul-Bunnell.

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PFC Responses of Mixed Tonsilar Lymphocyte

253

STIMULATION

4 Cultures: Effects of SRBC-Rosetting

Cells

No. of PFC/culture Target cells Mixed cultures

SRBC

BRBC

A” (-E) X B* (-E)’ A (-E) X B (+E)d A (+E) X B (-E)

25 320 710


15

A (-E) B (-E)

X A (+E) X B (+E)

20

230

AxB a A: cells from patient WL. b B: cells from patient JL. ’ (-E): cells deprived of E-rosetting cells. ‘(i-E): cells with E-rosetting cells.

Cellular requirement for stimulator cells in the PFC formation was studied. HTL of one individual were stimulated by HTL and PBL from another individual. These stimulator cells were separated into three populations, plastic-adherent, nylon-wool adherent, and nylon-wool-effluent cells. Each population was then used as a stimulator after treatment with mitomycin C. As seen in Table 5, plastic-adherent cells were the best, nylon-wool-effluent cells the intermediate, and nylon-wool-adherent cells (B-cell-rich population) the poorest stimulators. Effects of supernatants obtained from a mixed HTL culture on the PFC formation by another HTL stimulated by allogeneic HTL were studied. As seen in Table 6, the supernatant from the culture DM X JR was added to three different mixed HTL cultures. In all instances, including the same donor combination DM X JR, a significant increase in number of PFC against SRBC and BRBC was observed. The effect on PFC against SRBC appeared to be more pronounced than on PFC against BRBC. Similar results were obtained when another supernatant from a mixed culture of HTL and thymocytes was added to the three mixed HTL cultures. TABLE

5

PFC Responses of Tonsilar Lymphocytes Stimulated by Allogeneic Cells: Tonsilar Lymphocytes or Peripheral Blood Lymphocytes (II) No. of PFC against SRBC/culture Mitomycin C-treated stimulator cells

I

II

Non separated Plastic adherent Nylon wool adherent Nylon wool effluent

265 225 70 160

310 770 85 330

(I)

254

MORI ET AL. TABLE 6

PFC Responsesof Human Tonsilar Lymphocytes Stimulated by Allogeneic HTL: Effects of Supernatant from a Mixed Culture Number of PFC/culture in mixed culture of: DMXJR

Supernatant from mixed culture of DMXJR Medium

JR X BK

S

Bb

s

450

130

300

t10

110

80

CA X CD B

10
S

B

825

95

579

55

’ S: SRBC target cells. ’ B: BRBC target cells.

DISCUSSION In 1973, Hoffman et al. ( 17) reported that mixed cultures of HTL produced significant number of PFC against SRBC. However, their control cultures of HTL from one individual also produced a small number of PFC against SRBC. As noted in studies by other investigators (18, 19), and discussed in the previous communication (2), if fetal calf serum is added to the culture medium, positive results might be due to the stimulation of lymphocytes by highly immunogenic H-D antigen (20). This is why we used human AB serum instead of fetal calf serum throughout this investigation. In the present study, it was demonstrated that the vast majority of HTL specimens can produce significant number of PFC against SRBC and/or BRBC in response to various allogeneic lymphocytes. The PFC response of HTL apparently depended on an optimal dose of stimulator cells in the culture, since a IO-fold increase or decrease of the dose abolished almost completely the appearance of PFC. When HTL were stimulated with the optimal dose of allogeneic cells, PFC appeared 3-4 days after the initiation of the culture, reached the peak on the 6th day, and decreased in number between the 8th and 10th day. Specificities of antibodies secreted from the PFC were studied by inhibition tests. Results of these inhibition tests with solubilized heterophile antigens provided evidence that some of these antibodies were of F and others of H-D specificity. It does not appear likely that the PFC under study secreted P-B antibodies, since PB antigen failed to inhibit PFC formation. The possibility that some of the PFC might have secreted transplantation heterophile antibodies (3-6) remains to be explored. In the mixed HTL culture (two-way) experiments, it was shown that removal of E-rosetting (T) cells from both HTL populations resulted in over 90% reduction of PFC. Therefore, it may be concluded that T cells must be present at least in one of the HTL populations for formation of PFC. In analysis of stimulator cells in one-way experiments, it was observed that B cells were rather poor stimulators while plastic-adhering cells, presumably macrophages and monocytes, and nylonwool-effluent T cells were better stimulators.

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The fact that HTL but not PBL or spleen cells were able to produce a significant number of PFC indicates the possible in vivo priming of HTL by infectious agents. Results obtained in the experiments in which two HTL specimens were stimulated by six individual PBL seem also to support this possibility. Regardless of the donor of PBL, responses of HTL were quite similar in that the pattern of responses of these two HTL appeared to be predetermined: one HTL specimen (PS) formed PFC against SRBC only whereas another (GA) produced PFC against both SRBC and BRBC (Table 2). MerCtey et al. (21) have shown that human tonsils secrete small amounts of antibodies reacting with erythrocytes of foreign species such as sheep, rabbit, and chicken. Such primed HTL might have proliferated in vitro in responseto allogeneic stimulation, presumably because of the action of soluble nonspecific factor(s) which behave like polyclonal B-cell activators (22-27). The demonstration of enhancing effects of the supernatants from the mixed cultures seemed also to support this contention, since the supernatant obtained from an allogeneic combination exerted similar enhancing effects on the same and other combinations. It has been well established that allogeneic stimulation triggers murine primed B cells for antibody formation (28). Similarly, it has been reported that a nonspecific factor is released from human mixed lymphocyte cultures which enhances in vitro antibody formation of mice (9, 29). The marked inhibition of the PFC responseby a large number of stimulator cells could be explained by assuming that T cells in the stimulator, especially in the PBL may generate a killer T-ceil population which attacks proliferating PFC in the responder (30) or that suppressor T cells in the stimulator population may exert their effect on the PFC (26, 3 1). In initiating this study, we anticipated that stimulation of HTL by allogeneic cells will result in formation of antibodies directed against heterophile transplantation antigens (3-7). These antibodies were shown to be directed against antigens shared by human tissues and rat and bovine erythrocytes. The distribution of these specificities in human tissues appeared to reflect polymorphism in that antigens of various structures were expressedin different individuals. On the other hand, bovine (rat) erythrocytes contained the whole gamut of these specificities (3, 4). The present study indicated that in vitro allostimulation results in formation of heterophile antibodies of F and H-D variety rather than heterophile transplantation antibodies. Whereas the allocharacter of F antigen(s) was reported by some investigators (32) the allonature of H-D antigens remains to be elucidated. These questions will undoubtedly require further investigation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Mori, T., Kano, K., and Milgrom, F., Cell. Immunol. 34, 289, 1977. Mori, T., Kano, K., Merrick, J. M., and Milgrom, F., Cell. Immunol. 40, 28, 1978. Rapaport, F. T., Kano, K., and Milgrom, F., J. Clin. Invest. 47, 633, 1968. Kano, K., Loza, U., Gerbasi, J. R., and Milgrom, F., Transplantation 19, 20, 1975. Iwasaki, Y., Talmage, D., and Starzl, T. E., Transplantation 5, 191, 1967. McDonald, J. C., Transplantation 15, 116, 1973. McDonald, B. L., Hawkins, B. R., Dawkins, R. L., and Davey, M. G., VOX Sung. 33, 143, 1977. Terasaki, P. I.. McClelland, J., Park, M. S., and McCurdy, B., In “Manual of Tissue Typing Techniques” (J. G. Ray, D. B. Hare, P. D. Pedersen, and D. E. Kayhoe, Eds.), p. 67. Department

256

9. 10. 11. 12.

MORI ET AL.

of Health, Education and Welfare Publication, National Institutes of Health, Bethesda, Maryland, 1974. Rubin, A. S., and McDonald, A. B., immunology 30, 69, 1976. Jerne, N. K., and Nordin, A. A., Science 140, 405, 1963. Fuji, H., Schultz, R. T., and Milgrom, F., Proc. Sot. Exp. Biol. Med. 133, 180, 1970. Merrick, J. M., Schifferle, R., Zadarlik, K., Kano, K., and Milgrom, F., J. Suprumol. Struct. 6, 275, 1977.

13. Laine, R. A., Stellner, K., and Hakomori, S., In “Methods in Membrane Biology”, Vol. 2 (E. D. Korn, Ed.), Plenum, New York, 1974. 14. Mosier, D. E., Science 158, 1573, 1967. 15. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 16. Bach, J. F., Transplant. Rev. 16, 196, 1973. 17. Hoffman, M. K., Schmidt, P., and Oettgen, H. F., Nature (London) 243, 408, 1973. 18. Watanabe, T., Yoshizaki, K., Yagura, T., and Yamamura, Y., J. Zmmunol. 113, 608, 1974. 19. Baumohl, Z., Kellerhals, B., Stolp, W., and Lefkovits, I., Clin. Exp. Zmmunol. 28, 116, 1977. 20. Kasukawa, R., Kano, K., Bloom, M. L., and Milgrom, F., Clin. Exp. Immunof. 25, 122, 1976. 21. Merbtey, K., Koteles, G. J., and Elebes, E., Experientiu 28, 457, 1972. 22. Schimpl, A., and Wecker, E., Nature New Biol. 237, 15, 1972. 23. Feldmann, M., and Basten, A., J. Exp. Med. 136, 722, 1972. 24. Hunter, P., and Kettman, J. R., Proc. Nat. Acad. Sci. USA 71, 512, 1974. 25. Geha, R. S., Mudawwar, F., and Schneeberger, E., J. Exp. Med. 145, 1436, 1977. 26. Friedman, S. M., Breard, J. M., and Chess, L., J. Immunol. 117, 2021, 1976. 21. Aarden, L. A., Brunner, T. K., Cerottini, J-C., ef al., J. Immunol. 123, 2928, 1979. 28. Katz, D. H., Transplant. Rev. 12, 141, 1972. 29. Farrar, J. J., Koopman, W. J., and Fuller-Bonar, J., J. fmmunol. 119, 47, 1977. 30. Lefkovits, I., Eur. J. Immunol. 3, 397, 1973. 31. Saxon, A., Stevens, R. H., and Ashman, R. F., J. Immunol. 118, 1872, 1977. 32. Hakomori, S., Wong, S. M., and Young, W. W., Proc. Nat. Acad. Sci. USA 74, 3023, 1977.