Deletion of active human suppressor T lymphocytes from peripheral blood by sephadex G-10 filtration

Journal of Immunological Methods, 55 (1982) 327-336

327

Elsevier Biomedical Press

Deletion of Active Human Suppressor T Lymphocytes from Peripheral Blood by Sephadex G- 10 Filtration 1 Michael K. Hoffmann, Simon Pollack, Susan E. Krown and Robert S. Mittler * Memorial Sloan- Kettering Cancer Center, 12 75 York Avenue, New York, N Y 10021, and * Ortho Pharmaceutical Company, Raritan, N J, U.S.A.

(Received 26 March 1982, accepted 28 May 1982)

A method for antigen-specific generation of antibody-forming B cells in cultures of human peripheral blood mononuclear (PBM) cells based on the Mishell-Dutton system has recently been established in this laboratory. Comparing PBM cell cultures from healthy donors and from patients with advanced cancer we found the latter to be unresponsive in our assay. Passage of PBM cell suspension over Sephadex G-10 columns restored the response of patient PBM cells to normal levels. The cell population trapped on the column can be recovered and its inhibitory potential demonstrated by its graded addition to cells eluted from the column. The cell responsible for inhibition is sensitive to treatment with OKT8 antibody and complement, indicating its T cell nature. Passage of PBM cells from healthy individuals did not alter antibody responses substantially but made the activation requirements less stringent. Key words: antibody production in vitro - - cell surface phenotypes - - cancer patients

Introduction A method for antigen-specific sensitization of human peripheral blood monon u c l e a r ( P B M ) cells h a s r e c e n t l y b e e n d e v e l o p e d i n t h i s l a b o r a t o r y ( H o f f m a n n , 1 9 8 0 a ; R e i s n e r et al., 1980; P a h w a et al., 1981a, b). T h e m e t h o d is b a s e d o n t h e t e c h n i q u e d e s c r i b e d b y M i s h e l l a n d D u t t o n ( M i s h e l l a n d D u t t o n , 1967) f o r t h e s e n s i t i z a t i o n o f m u r i n e s p l e e n cells. H u m a n P B M cell c u l t u r e s a r e i m m u n i z e d w i t h

1 This investigation was supported by a grant from the American Cancer Society, ACS-IM-219, by an American Cancer Society Junior Faculty Fellowship, and by the Lazar Fellowship Israel Scholar Exchange Program. Abbreviations: BSS, balanced salt solution; CMDM, complete Mishell-Dutton medium; FCS, fetal calf serum; PBM, peripheral blood mononuclear cells; PFC, plaque-forming cells; SRBC, sheep erythrocytes; Staph A, Staphylococcus aureus, Cowan I strain. 0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

328 red blood cell bound antigen and the generation of antibody-forming cells is determined 6-7 days later using a hemolytic plaque assay (Mishell and Dutton, 1967). Using this method, it has been shown that T cells participate both as helper cells and as suppressor cells in the regulation of antibody formation by human peripheral blood B lymphocytes (Hoffmann, 1980a). We have used this method to study humoral immune function in patients with advanced malignant melanoma and have found a significant reduction in their in vitro antibody responses. Based on preliminary experiments in which the addition of PBM cells from patients was shown to decrease the in vitro antibody response of PBM cells from normal individuals, it was suggested that suppressor cells might account for the abnormal antibody responses of cancer patients. We have, therefore, investigated the role of suppressor cells in regulating in vitro antibody production by PBM cells from cancer patients and normal subjects using both monoclonal antisera to human T cell antigens and passage of the cells over Sephadex G-10 columns. The latter technique, which had previously been shown to remove suppressor T cells from suspensions of mouse lymphoid cells (Pickel and Hoffmann, 1977a, b) was found to be an effective, simple and reproducible method for removing suppressor cells from human PBM cell suspensions.

Materials and Methods

Reagents Sheep erythrocytes (SRBC) were obtained from the Colorado Serum Company (Denver, CO). Staphylococcus aureus Cowan strain I (Staph A (American type culture collection, Rockville, MD), no. 1285) was cultured and inactivated as described (Hoffmann, 1980a). Sephadex G-10 was purchased from Pharmacia Fine Chemicals (Piscataway, N J). Sodium-metrizoate/Ficoll (Lymphoprep) was obtained from Nyegaard (Oslo), and fetal calf serum (FCS) (lot no. 94055) from Microbiological Associates (Walkerville, MD). Monoclonal OKT8 antibody was produced by Ortho Pharmaceutical Co. (Raritan, N J). Preparation of PBM Heparinized venous blood was obtained from 20 patients with stage III or stage IV malignant melanoma and 23 normal controls. Only those patients were used in these experiments who had not received treatment for at least 6 weeks. Peripheral blood mononuclear (PBM) cells were isolated from the blood by the standard Ficoll-Metrizoate density centrifugation method, washed twice with RPMI 1640 medium, and resuspended in complete Mishell-Dutton medium (CMDM) (Mishell and Dutton, 1967). Preparation of Sephadex G-IO columns Sephadex G-10 columns were prepared as described by Ly and Mishell (1974) with slight modifications. Sephadex G-10 was washed in distilled water and saline, resuspended in saline, autoclaved (100°C) and stored at room temperature. Columns

329 were prepared by gently pipetting 5 ml of the Sephadex-saline slurry into a 10 ml plastic syringe barrel (Becton-Dickinson no. 5604) with the needle port blocked by a plastic bead. The saline was allowed to run out, the column washed with 10 ml of warm (42°C) balanced salt solution (BSS) and incubated at 37°C for 1 h. Before passing PBM cells, the columns were washed once with 5 ml of warm (42°C) BSS, and then with 5 ml of BSS, containing 20% FCS at 45°C. Cell cultures PBM cell cultures were performed as described previously (Hoffmann, 1980a). Briefly, 0.1 ml volumes consisting of serial log 2 dilutions of PBM ranging from 5-1.25 x 10 6 cells/ml were plated in flat bottom microtiter plates (Costar Data Packaging, Cambridge, MA, no. 3596) and immunized with 5 x 10 6 SRBC (0.03% final concentration). Cultures also contained inactivated Staphylococcus aureus Cowan strain I (0.003% final concentration) as a selective B cell mitogen (Pahwa et al., 1981 a) and a supernatant fluid of lipopolysaccharide-stimulated adherent human PBM cells (Finelt and Hoffmann, 1979), as a source of Interleukin-1 (3% final concentration). After 16-20 h incubation, pooled human AB serum was added to the cultures at a final concentration of 10%. SRBC-absorbed sera were not used in these experiments because of the possibility of dissolved antigen obscuring antigen-negative controls. Cultures were fed every other day with 10 #1 of nutritional cocktail (Mishell and Dutton, 1967) and harvested on day 6 or 7. Cells were passed over Sephadex G-10 columns prior to culture, as follows: 1 ml of a cell suspension containing 2 - 5 x 10 v PBM cells/ml in R P M I 1640 medium supplemented with 20% FCS was added to the column and eluted (at 1 g with flow rate of 1-1.5 m l / m i n ) with 5 ml of warm (37°C) BSS containing 5% FCS. Cells retained on the column were recovered by vigorously shaking the Sephadex beads in the syringe barrel and then allowing the Sephadex beads to settle by gravity. Cells remaining in the supernatant and cells passed through the column were washed twice and resuspended in medium. Of the cells originally applied to the column, 50-60% pass through into the initial eluate; cells subsequently recovered from the Sephadex column by shaking constitute 10% of the original inoculum. All cultures were incubated for 6 - 7 days on a rocker platform (6 cycles/min) at 37°C in an atmosphere of 7% 02, 10% CO 2, 83% N 2 and 100% humidity. Plaque-forming cell assays At the end of the culture period, cells were washed in R P M I 1640 medium containing 5% FCS, resuspended in BSS, and the antibody response assayed using a Jerne hemolytic plaque assay as modified by Mishell and Dutton (1967). Anti-SRBC plaque-forming cells (PFC) were enumerated as described (Mishell and Dutton, 1967) and expressed as the number of PFC per 10 6 cultured cells. The number of anti-SRBC PFC in cultures incubated without antigen was never greater than 10% of that seen in antigen-stimulated cultures of either normal control or patient PBM cells.

330

Identification of monocytes M o n o c y t e s were identified b y ingestion of latex particles, 0.801/~m in d i a m e t e r as d e s c r i b e d p r e v i o u s l y ( K o z i n e r et al., 1977).

Complement-dependent lysis of OKT8-positive T cells T h r e e times 107 P B M cells were i n c u b a t e d for 30 min on ice in 1 ml of O K T 8 a n t i b o d y (0.04 m g / m l ) . N o n - t o x i c r a b b i t serum as a source of c o m p l e m e n t (0.25 ml) was then a d d e d a n d the mixture i n c u b a t e d for 45 min at 37°C. Cells were w a s h e d 3 times in R P M I 1640 m e d i u m c o n t a i n i n g 1% F C S a n d r e s u s p e n d e d in C M D M . G e n e r a l l y , less than 3% of the cells were O K T 8 - p o s i t i v e after t r e a t m e n t with antiserum and complement.

Results

Generation of antibody-forming cells in cultures of PBM cells from cancer patients P B M cells o b t a i n e d from 20 p a t i e n t s with a d v a n c e d m e l a n o m a a n d from 23 h e a l t h y subjects were c u l t u r e d a n d i m m u n i z e d with sheep e r y t h r o c y t e s ( S R B C ) ( H o f f m a n n , 1980a). A n t i b o d y f o r m i n g B cells g e n e r a t e d in culture were e n u m e r a t e d 6 d a y s later in h e m o l y t i c p l a q u e assays (Mishell a n d D u t t o n , 1967). P B M cells o b t a i n e d f r o m p a t i e n t s either failed to generate P F C against S R B C in culture or showed low responses as c o m p a r e d to P B M cells from h e a l t h y d o n o r s (Fig. 1). W h e n

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Fig. 1. Effect of Sephadex G-10 column filtration on the PFC response in cultures of PBM cells from healthy donors and from cancer patients. PBM cells were cultured unseparated (O) or following passage over Sephadex G-10 columns (O) and PFC formation tested 6 days later. All cultures were immunized with SRBC.

331

PBM cell suspensions from normal subjects were passed over Sephadex G-10 columns prior to culture, there was no significant change in the PFC response. By contrast, when PBM cell suspensions from cancer patients were passed over Sephadex G-10 columns, the eluted cells showed normal anti-SRBC PFC responses (Fig. 1). These findings indicate that antigen reactive B cells and helper T cells are adequately represented in the peripheral blood of cancer patients and that the failure of unseparated cells to mount PFC responses must be attributed to suppressor cells that can be trapped on Sephadex G-10 columns. The routine addition of monocyte conditioned medium containing Interleukin-1 (Hoffmann and Watson, 1979; Hoffmann, 1980b) to the PBM cell cultures insured the presence of optimal monocyte helper function even in cultures depleted of monocytes after Sephadex G-10 filtration (Ly and Mishell, 1974).

Characterization of suppressor cells from cancer patients Sephadex G-10 filtration was originally described as a method to remove macrophages and monocytes from lymphoid cell suspensions (Ly and Mishell, 1974) but was subsequently found also to be an effective technique for the removal of suppressor T cells from murine spleen cell suspensions (Pickel and Hoffmann,

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Fig. 2. Column-retained suppressor cells are eliminated by treatment with OKT8 antibody and complement. PBM cells retained on Sephadex G-10 columns were added in graded numbers (as indicated) to a constant number of 2.5 × 105 per culture to PBM cells eluted from the column. PFC formation was determined 6 days later from pools of 8 cultures each. Column retained cells were treated with NMS and complement (closed symbols) or OKT8 antibody and complement (open symbols). Data obtained with PBM cells from 3 cancer patients are shown.

332 TABLE I PHENOTYPIC CHARACTERISTICS OF UNSEPARATED PBM CELLS COMPARED TO CELLS ELUTED FROM OR RETAINED ON SEPHADEX G-10 COLUMNS Cell surface antigenic markers were analyzed by flow microfluorometry with an Ortho FC 200-4800 ASO cytofluorograph (Ortho Instruments, Westwood, MA). lmmunofluorescence staining of the cells was performed in a 2-step technique (Koziner et al., 1977) involving monoclonal antibody reagent in the first step and fluorescein-conjugated F(ab')2 goat antibody to mouse immunoglobulin in the second step. OKT4 antibody (designating inducer/helper T cells (Reinherz et al., 1979)) and OKT8 antibody (designating suppressor/killer T cells (Reinherz et al., 1980)) was prepared by Ortho Pharmaceutical Co. (Raritan, NJ). Monocytes were identified by ingestion of latex particles (0.801 #m in diameter) as described (Koziner et al., 1977). Donor

Control

Patient 1

Patient 2

Treatment

Unseparated Seph. eluted Seph. retained Unseparated Seph. eluted Seph. retained Unseparated Seph. eluted Seph. retained

Cells positive for marker (%) OKT4

OKT8

Latex

44 41 23 24 26 15 32 44 28

23 25 20 33 28 30 26 18 24

13 4 27 26 2 25 14 4 19

Anti-SRBC PFC/IO 6 cells

ND ND ND 27 688 4 0 467 0

1977a, b). It was possible, therefore, that the peripheral blood cell which was responsible for the d i m i n i s h e d a n t i - S R B C P F C responses observed in cultures of P B M cells from m e l a n o m a patients, a n d which was retained o n Sephadex G-10 c o l u m n s , was either a m o n o c y t e or a T cell. Preliminary experiments (data not shown) suggested that suppressor T cells were involved, since P F C responses could be increased if P B M cells were treated with a n t i - O K T 8 a n t i b o d y a n d c o m p l e m e n t prior to culture. These experiments were consistent with the observation that e l i m i n a t i o n of O K T 8 - p o s i t i v e cells eliminates suppression mediated by h u m a n T cells (Reinherz et al., 1980; T h o m a s et al., 1981) b u t they did not permit the c o n c l u s i o n that cells retained o n Sephadex G-10 were themselves O K T 8 - p o s i t i v e suppressor T lymphocytes rather t h a n being required for the activation of O K T 8 positive T cells. T o test these alternatives, cells that were retained on Sephadex G-10 c o l u m n s were collected a n d added back, either u n t r e a t e d or treated with a n t i - O K T 8 a n t i b o d y a n d c o m p l e m e n t , to the PBM cells that were eluted from the column. U n t r e a t e d cells recovered from the c o l u m n were f o u n d to inhibit the P F C response of cells eluted from the c o l u m n (Fig. 2). However, if the recovered cells were first treated with a n t i - O K T 8 a n t i b o d y and c o m p l e m e n t prior to their a d d i t i o n to cultures of eluted cells, the P F C responses were restored to n o r m a l (Fig. 2). These data indicate that the S e p h a d e x - a d h e r e n t suppressor cell in the PBM cell suspension tested is a n O K T 8 - p o s i t i v e T cell.

333 TABLE II EFFECT OF EARLY AND DELAYED ADDITION OF HUMAN SERUM ON ANTIBODY PRODUCTION IN CULTURES OF UNSEPARATED AND SEPHADEX G-10 TREATED HUMAN PBM Each value represents the response of eight pooled cultures. PBM were obtained from healthy donors. PBM

Exp. 1 Exp. 2

Anti-SRBC PFC/106 cells

Unseparated SephadexG-10 Unseparated SephadexG-10

HS a day 0

HS day 1

116 1200 8 86

880 940 85 162

Human serum 10%.

P h e n o t y p i c a n a l y s i s w a s m a d e o f u n s e p a r a t e d P B M cells a n d o f cells e l u t e d f r o m the Sephadex G-10

columns. No

consistent differences were seen between the

s e p a r a t e d a n d u n s e p a r a t e d cell p o p u l a t i o n s w i t h r e g a r d t o t h e p r o p o r t i o n o f O K T 8 p o s i t i v e cells. M o n o c y t e s w e r e e f f e c t i v e l y d e p l e t e d a f t e r p a s s a g e o v e r t h e S e p h a d e x G - 1 0 c o l u m n s ( T a b l e I). It w o u l d a p p e a r , t h e r e f o r e , t h a t O K T 8 - p o s i t i v e cells p e r se

TABLE III GENERATION OF ANTIBODY FORMING CELLS IN CULTURES OF SEPHADEX G-10TREATED HUMAN PBM IN THE ABSENCE OF Staphylococcus aureus BACTERIA (Staph A) Human serum was added after 20 h of culture. PBM were obtained from healthy donors. PBM

Anti-SRBC PFC/106 cells

Staph A Exp. 1 Unseparated Unseparated Seph G-10 Seph G- 10 Exp. 2 Unseparated Unseparated Seph G-10 Seph G-10 Exp. 3 Unseparated Unseparated Seph G-10 Seph G-10

SRBC

No Ag

+ +

860 0 520

42 0 40

-

185

0

+ + -

786 0 1240 215

68 0 123 0

+ + -

329 0 679 170

29 0 76 0

334 are not responsible for inhibition of PFC responses, but rather that a subpopulation of OKT8-positive T cells which is retained on the Sephadex G-10 columns is responsible.

Effect of Sephadex G-IO filtration on the PFC response of PBM cells obtained from healthy donors Column separation of PBM cells from healthy subjects did not significantly alter their PFC responses in vitro. This was unexpected because evidence for suppressor cell activity had been observed in cultures of PBM cells from healthy donors (Hoffmann, 1980a; Reisner et al., 1980; Pahwa, 1981 a, b). In developing our method for antigen-specific sensitization of human PBM cells, we had previously believed that the difficulty of inducing antibody production in cultures of human peripheral blood lymphocytes was attributable to a disproportionate representation of suppressor cells in PBM cell suspensions (Hoffmann, 1980a). In order to overcome their inhibitory influence on the B cell response, we added a B cell mitogen (Staphylococcus aureus, Cowan I strain) to the cultures, with the rationale of stimulating the B cells with both an antigen and a mitogen before suppressive influences were fully established. We found it necessary to add human serum after 16-20 h rather than at the initiation of the culture period, presumably also to prevent activation of suppressor cells (Hoffmann, 1980a). Staphylococcus aureus was, in our experience, an essential additive to the cultures, while the delayed addition of human serum merely improved PFC responses (often considerably). With the Sephadex G-10 filtration method established as a method to remove suppressor cells from lymphoid cell populations we were able to test the hypothesis that these measures were, in fact, serving to overcome inhibitory influences of suppressor cells. We found, indeed, that h u m a n PBM cells eluted from Sephadex G-10 columns no longer depended upon the addition of Staph A for the generation of antibody-f0rming cells and that it was no longer necessary to delay the addition of human serum. The effect of early and delayed addition of human serum is demonstrated in Table II which shows 2 experiments representing 2 different magnitudes of antibody response. Table III shows the results of 3 experiments in which PBM cells were cultured in the presence or the absence of Staph A. Antibody-producing cells were generated in the absence of Staph A in cultures of Sephadex G-10-treated PBM cells but not in cultures of unseparated PBM cells. Antigen-independent (polyclonal) PFC responses were observed only in Staph A-treated cultures. Cultures of Sephadex G-10-treated cells responded still better in the presence of Staph A than in its absence. However, the significance of this experiment lies in the demonstration that human PBM cells can mount antigen-specific PFC responses in the absence of a polyclonal stimulus.

Discussion

Our studies establish Sephadex G-IO filtration as a simple method capable of altering the cellular composition of human peripheral blood mononuclear cells and their ability to generate antibody-formin~ cells in vitro. These data demonstrate that

335

passage of human PBM cells over Sephadex G-10 columns eliminates cells which exert a suppressive influence on PFC responses in vitro. Using defined antibody to cell surface antigens we have shown that the column retains OKT8-positive suppressor T lymphocytes. This finding is consistent with previous observations made with Sephadex G-10 columns and murine suppressor T lymphocytes (Pickel and Hoffmann, 1977a, b). Sephadex G-10 filtration can be a useful tool in studies concerned with the regulation of humoral immunity in man and in clinical studies aimed at monitoring the immune status of patients. While the data presented herein concern patients with malignant melanoma, similar results have been obtained in preliminary studies in patients with other types of cancers. In general, unseparated PBM cells from cancer patients show decreased anti-SRBC PFC responses which are restored to normal following Sephadex G-10 filtration. Similar studies are now in progress in patients with primary immunodeficiency and in patients who received bone marrow transplants. The PBM cells from the majority of these patients are also unresponsive in the PFC assay but become responsive when passed over Sephadex G-10 columns. However, in these patients, cell surface phenotypic analysis has revealed that suppressive activity cannot always be attributed to T cells, but may, in some cases, be attributable to T cells and monocytes or, on occasion, exclusively to monocytes (S. Pollack, manuscript in preparation). The results presented in this paper support our contention that suppressor cells exert a significant influence on the generation of antibody-forming cells in cultures of human PBM cells. The addition of a B cell mitogen and the delayed addition of human serum to cultures, initially intended as measures to allow B cells to overcome the influence of suppressor cells, are no longer essential when PBM cells are cultured in the absence of Sephadex G-10 adherent cells. The absolute requirement for a B cell mitogen in our culture system has been subject to criticism. It has been argued that Staph A is a polyclonal B cell activator, and it was unclear to what extent the measured PFC response reflected antigen-specific or polyclonal antibody formation. This argument is no longer relevant when PBM cells are cultured under the conditions described herein. After removal of suppressor cells, antigen alone induces specific antibody formation in cultures of human PBM cells without a measurable polyclonal antibody response. Polyclonal antibody formation occurs in cultures that contain Staph A. However, the polyclonal PFC response is one-tenth that of the antigen-specific PFC response and thus still satisfies the same criteria of specificity accepted for the Mishell-Dutton system in the mouse. The role of human serum in the generation of human antibody-forming cells must also be considered with respect to the activation of suppressor cells. We have shown that delayed addition of human serum to PBM cell cultures was more effective in supporting PFC responses than its immediate addition. This effect was ascribed to the activation of suppressor cells, an hypothesis which is supported by our finding that PBM cells depleted of suppressor cells do not require delayed addition of serum for optimal PFC responses, and which is consistent with observations made with cultured murine spleen cells indicating that suppressor T cells are effective only if present in the early phase of the culture (Pickel and Hoffmann, 1977a). We interpret

336 these findings to suggest that inactive suppressor cells become activated in vitro by serum factors, a n d that their activation affects the g e n e r a t i o n of PFC, particularly w h e n this occurs early in the i n c u b a t i o n period. The relationship between the p r e s u m a b l y n o n - a c t i v a t e d suppressor cells in the blood of healthy subjects a n d the p r e s u m a b l y activated suppressor cells f o u n d in the b l o o d of cancer patients (both retained o n Sephadex G-10 columns) is not clear at present, b u t one may speculate that serum factors may be involved in the control of suppressor cells in vivo as well as in vitro. The m e t h o d we have described for analysis of factors i n f l u e n c i n g the h u m o r a l i m m u n e response in m a n should provide suitable means to clarify this question. Of note is the finding that removal of Sephadex G-10 adherent O K T 8 - p o s i t i v e T cells, while resulting in complete r e c o n s t i t u t i o n of the P F C response, does not n o t i c e a b l y affect the p r o p o r t i o n of O K T 8 - p o s i t i v e cells in the c o l u m n eluate. Since l y m p h o i d cells are i n c u b a t e d on the c o l u m n s for only a short period, requiring only a few m i n u t e s for elution, we conclude that the suppressor T cells are retained in Sephadex G-10 columns, along with macrophages a n d p l a s m a cells (Ly a n d Mishell, 1974), as a c o n s e q u e n c e of their size. Small O K T 8 - p o s i t i v e lymphocytes a p p a r e n t l y pass through the c o l u m n freely a n d do not exhibit suppressor cell f u n c t i o n in the P F C assay. The active suppressor cell may thus be described as a large O K T 8 - p o s i tive T cell or blast. If these cells are, in fact, being retained on the c o l u m n s on the basis of their size, it would suggest that d e t e r m i n a t i o n of the fraction of O K T 8 - p o s i tive cells alone would be of little value as a measure of potential suppressor cell activity. It would follow that d e t e r m i n a t i o n of the p r o p o r t i o n of O K T 8 - p o s i t i v e T cell blasts is more likely to be useful for the assessment of peripheral blood suppressor T cell activity than would be simple e n u m e r a t i o n of the O K T 8 - p o s i t i v e cell set.

References Finelt, M. and M.K. Hoffmann, 1979, Clin. Immunol. Immunopathol. 12, 281. Hoffmann, M.K., 1980a, Proc. Natl. Acad. Sci. U.S.A. 77, 1139. Hoffmann, M.K., 1980b, J. Immunol. 125, 2076. Hoffmann, M.K. and J. Watson, 1979, J. Immunol. 4, 1371. Koziner, B., S. McKenzie, D. Straus, B. Clarkson, R.A. Good and F.P. Siegal, 1977, Blood 49, 895. Kung, P.C. and G. Goldstein, 1980, Vox Sang. 39, 121. Ly, I.A. and R.I. Mishell, 1974, J. Immunol. Methods 5, 239. Mishell, R.I. and R.W. Dutton, 1967, J. Exp. Med. 126, 423. Pahwa, S.G., R.A. Good and M.K. Hoffmann, 1981a, J. Clin. lmmunol. 1, 101. Pahwa, S.G., R.N. Pahwa and R.A. Good, 1981b, J. Clin. Invest. 67, 1094. Pickel, K. and M.K. Hoffmann, 1977a, J. Immunol. 118, 653. Pickel, K. and M.K. Hoffmann, 1977b, J. Exp. Med. 145, 1169. Reinherz, E.L., P.C. Kung, G. Goldstein and S.F. Schlossman, 1979, J. Immunol. 123, 2894. Reinherz, E.L., P.C. Kung, G. Goldstein and S.F. Schlossman, 1980, J. Immunol. 124, 1301. Reisner, Y., S.G. Pahwa, J.W. Chiao, N. Sharon, R.L. Evans and R.A. Good, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 6778. Schurrman, R.K.B., E.W. Gelfand and H.M. Dosch, 1980, J. Immunol. 125, 820. Thomas, Y., J. Sosman, O. Irigoyen, S.M. Friedman, P.C. Kung, G. Goldstein and L. Chess, 1981, J. Immunol. 125, 2402.