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Mechanisms Involved in the Antibody-Mediated Suppression of Tuberculin-Type Delayed Hypersensitivity

II. The Sensitivity of Tolerance Induction to Cyclophosphamide Pretreatment and Splenectomy, and the Demonstration of Active Suppression GEORGE S. DOUVAS AND ALFRED J. CROWLE
Department of Microbiology and Immunology, University of Colorado Health Sciences Center, and Webb- Waring Lung Institute, Denver, Colorado 80602 Received June 26, 1980; accepted July 17. 1980 The induction of tuberculin-type delayed hypersensitivity, as measured by skin test, can be specifically inhibited by administration of antibody during sensitization. The cellular mechanisms involved in this tolerance were investigated in CAF, mice, using chicken conalbumin as antigen. Tolerance was prevented when mice were treated with cyclophosphamide 2 days before sensitization and suppression. However, it was not affected by splenectomy 7 or 21 days before sensitization. This tolerance could be transferred to normal CAF, mice with spleen cells, but not with thymocytes, when taken from donor mice 21 to 28 days after sensitization and tolerance induction. Production of these cells in the donors required both antibody and antigen. The cells responsible for the transfer were B cells, as shown by their sensitivity to rabbit anti-mouse-immunoglobulin serum and complement. In addition to B cells, serum from tolerant mice also could transfer suppression at 21 to 28 days. We conclude that sensitizing mice, and treating them with specific immunosuppressive antiserum, induces the recipients to make suppressor B cells and suppressive humoral factors, which are involved in arresting the induction of tuberculin-type delayed hypersensitivity.

INTRODUCTION When mice are injected with purified protein antigens in an efficient adjuvant, they develop a slowly arising, long-lived type of delayed hypersensitivity (DH) (1). If mice are injected at the time of sensitization with hyperimmune mouse antiserum, its IgG, subfraction, or hyperimmune guinea pig antiserum or its IgGz subfraction, the induction of this type of DH is specifically supressed (2-4). The precise mechanism of this suppression is unknown. Increasing evidence suggests antigen blockade is not involved because: (a) the capacity of an antiserum to induce
This work was supported by a grant from the John A. Hartford Foundation, and Grants 5T32AI07035, 5ROlAI09867, and 5ROlAG00008 from the National Institutes of Health. Abbreviations used: DH, delayed hypersensitivity; AH, Arthus hypersensitivity; Cy, cyclophosphamide; CA, chicken conalbumin; w/o, water-in-oil emulsion; PPB, physiologic phosphate buffer; PB, phosphate buffer; anti-BA-8, rabbit anti-mouse brain-associated-& anti-MIg, rabbit anti-mouse immunoglobulin; NRS, normal rabbit serum; C, guinea pig complement; HBSS, Hanks balanced salt solution; As, antiserum.

0008-874918l/040302-10502.00/O
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reswvcd.

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tolerance does not correlate infallibly with its ability to precipitate or agglutinate antigen (3), (b) F(ab), fragments cannot tolerize (4), (c) tolerance induction is not affected by the amount of antigen used to sensitize, (d) antigen-antibody complexes can suppress,3and (e) tolerance does not inhibit antigen-specific proliferative responsesof lymph node cells.3 Suppression is not, at least directly, mediated by anti-idiotype antibodies because the immunosuppressive antibodies can be adsorbed to and eluted from insolubilized antigen (3). Another mechanism by which antibodies could tolerize is by inducing the production of suppressor cells, a form of immunoregulation now well characterized (5-8). We have used several approaches to investigate this possibility. One, popular in recent years, is to use the drug cyclophosphamide (Cy). Cy, an alkylating agent, has been shown to eliminate the precursors of suppressor cells in several experimental systems (9-11). A second approach has been to test the effects of splenectomy on tolerogenesis, since the spleen has been shown to be a major source of suppressor cells (12, 13). The third and most direct approach has been to see whether suppression is transferrable with cells from tolerant mice. We report here that the tolerance induced by antibodies is Cy sensitive, is not affected by splenectomy, and is transferrable with B cells and serum from tolerant mice. MATERIALS AND METHODS

Animals. Eight- to ten-week-old female (BALB/c X A/J) F, hybrid mice (CAF,) were purchased from Jackson Laboratories (Bar Harbor, Maine), and outbread CF-1 female mice of the same age from Carworth Farms (Portage, Mich.). New Zealand White rabbits were purchased from the Sedalia Hills Rabbitry (Sedalia, Colo.). Antigens. Chicken conalbumin (CA) type I was purchased from the Sigma Chemical Company (St. Louis, MO.). Sensitization, tolerance induction, and their measurement. CAF, females were sensitized by subcutaneous (SC) injection in the inguinal region on Day 0 with 20 pg CA in 0.1 ml of water-in-oil emulsion (w/o). This Freunds incomplete adjuvant was made as described by Crowle and Hu (14). To induce tolerance, mice sensitized on Day 0 were injected intraperitoneally (ip) on Days O-4 with 0. l-ml volumes of hyperimmune antiserum to CA, diluted in physiologic phosphate buffer (PPB) to a final antigen-binding capacity of either 559 or 1118 pg/ml. Sensitization was measured on Days 14, 28, and 42 with intradermal injections of 0.02 ml PPB, containing 20 pg of antigen. Such skin tests were made on alternating shaved flanks, and inflamation diameters were measured at 3 and 24 hr using vernier calipers. Three- and twenty-four-hour readings represent Arthus hypersensitivity (AH) and DH, respectively (1). Although both reactions were measured in these experiments, AH will not be discussed in this paper. It is usual with this kind of test that mice react either measurably @S-mm reaction diameter), or not at all (nothing palpable), creating a bimodal distribution of values (15). For this reason, results have been expressed as percentage of mice reacting to antigen on indicated days, instead of mean reaction diameter.
3 Manuscript submitted for publication.

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Antisera. Immunosuppressive antiserum to CA was produced using a procedure described previously.3 CF-I mice were sensitized as described above using 100 pg CA instead of 20 pg. They were then skin tested at 14 and 28 days to verify the development of good AH and DH. Fourteen to twenty-one days later, they were injected ip on 4 succeeding days with O.l-, l.O-, lO.O-, and 20-mg quantities of CA in O.l-ml (0.1-10 mg) or 0.2-ml (20 mg) volumes of PPB. Six days after the 20.0mg injection they were bled out and their sera pooled and stored at -20C until needed. This intensive regimen of antigen injection is necessary to secure antiserum with high titers of immunosuppressive antibodies (16). Rabbit anti-brain-associated-8 serum (anti-BA-8) was prepared using the procedure described by Golub ( 17) with the exception that boostings were done using incomplete instead of complete Freunds adjuvant. The procedure used to purify mouse immunoglobulins for making rabbit antimouse-immunoglobulin serum (anti-MIg) has been described.3 Briefly, mouse immunoglobulins were purified from hyperimmune serum by preparatory zone electrophoresis using 2% agar in barbital-acetate buffer, pH 8.2. Immunoglobulins were eluted from sections cut from the resulting gel by freezing and thawing. Anti-MIg was produced by injecting 0.17 mg of immunoglobulin protein into rabbits, in 2 ml w/o, 0.5 ml portions being injected SCat four different sites, two over the rear legs and two over opposite shoulders. Twenty-one days after sensitization, the rabbits were boosted with 0.4-ml volumes of the same emulsion injected SC.Twelve, thirteen, and fourteen days after boosting, rabbits received 0.1, 1, and 2 ml, iv of purified immunoglobulin containing approximately 2.6 mg/ml of protein. The rabbits were bled for antiserum 8 and 19 days after the last injection. Before use, i ml of anti-BA-8 serum was absorbed twice for 30 min at 4C each time with CAF, bone marrow cells from two femurs and two tibias, and 0.5 ml packed, heparinized, red blood cells. Anti-MIg serum was similarly absorbed with 0.5 ml packed, heparinized, red blood cells, and cells prepared from one thymus. One milliliter normal rabbit serum (NRS) was absorbed two times with 0.5 ml packed, heparinized, red blood cells, and cells prepared from one thymus and one spleen. Spleen cells and thymocytes were prepared by teasing organs apart in Hanks balanced salt solution (Microbiological Associates, Walkersville, Md.) (HBSS) and washing twice by centrifugation (700 g, 7 min) using the same buffer. Anti-BA-6 serum, anti-MIg serum, and NRS treatment of cells. Spleen cells or thymocytes were reacted with 1.0 ml of one-to-five or one-to-ten dilutions of NRS, anti-BA-0, or anti-MIg serum per lo* cells for 30 min at 4C washed once in HBSS, and incubated with 1.0 ml of a one-to-five or one-to-ten dilution of guinea pig serum complement (C) per lo8 cells for 30 min in a 37C waterbath. Cells were then washed twice in HBSS. Cell killing was determined using the trypan blueeeosin dye exclusion test (18). Cyclophosphamide treatment. Cyclophosphamide (Mead Johnson and Co., Evansville, Ind.) was injected iv at 150 mg/kg of body wt in distilled water. Splenectomy. Mice were splenectomized and sham splenectomized under sodium pentobarbital (Fort Dodge Laboratories, Fort Dodge, Iowa) anesthesia. Radiolabeling. CA was labeled with 125Iusing a modification of the chloramineT method (described in Reference 19). Na 1 (0.1 ml of 20 mCi/ml) (New England Nuclear, Boston, Mass.) was buffered with 0.1 ml of 0.25 M phosphate buffer (PB), pH 7.5. To this were added in rapid succession, with continuous

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agitation: 0.01 ml of a 0.5-g/ml solution of CA in 0.5 M PB, pH 7.5; 0.01 ml of a 5-mg/ml chloramine-T solution in 0.05 M PB, pH 7.5; and 0.1 ml of a 1.2-mg/ ml sodium metabisulfite solution in 0.05 M PB, pH 7.5. The volume of the solution was then made up to 1.0 ml with a 2.0 mg/ml sodium iodide solution in 0.05 M PB, pH 7.5. The radioiodinated CA was then extensively dialyzed against PPB. Specific activity of the radioiodinated CA was 28 &i/mg protein. CA was examined for damage caused by the radioiodination procedure using the method described in (19). Results showed that CA was not damaged during radioiodination. Determination of antigen-binding capacity. Antigen-binding capacity (ABC) of total serum was determined using a double-antibody method (20). Volumes (0.025 ml) of CF-1 NMS were added as carrier to 12 X 75-mm polypropylene culture tubes. This amount of normal serum was determined by titration to give maximum precipitation. To this were added 12-pg quantities of CA containing trace amounts of radiolabeled CA, in 0.012 ml PPB, and 0.2-ml dilutions of antiserum or antibody in PPB. The mixture was then incubated for 5 days at 4C. At the end of 5 days 0.213 ml PPB and 0.05 ml anti-MIg were added and the tubes were allowed to stand another 24448 hr at 4C. The concentration of rabbit antiserum to use for maximum precipitation had been determined by previous titration. The tubes were then counted for total label. The precipitate was spun down by centrifugation at 17,000g for 12 min, washed twice with PPB, and counted to determine the percentage of antigen bound. Statistics. P values for the data were determined using the x2 test (21). EXPERIMENTS
Effects of Cyclophosphamide Delayed Hypersensitivity

AND RESULTS
on Antibody-Mediated Inhibition of

Pretreatment

To determine whether Cy affects the expression of tolerance, groups of mice were pretreated with Cy and later sensitized, or sensitized and suppressed with antiserum to CA. The results of this experiment (Fig. 1) show that Cy pretreatment abrogated the antiserum-mediated suppression for both 28-day, and more markedly, 42-day skin tests.
Role of the Spleen in Antibody-Mediated Suppression of Delayed Hypersensitivity

We next investigated the role of the spleen in both the development of delayed hypersensitivity in mice, and suppression of this hypersensitivity by antibodies. For these experiments mice were either splenectomized or sham splenectomized 7 to 21 days before sensitization. On Day 0 mice were sensitized, or sensitized and suppressed with antiserum, and skin tested as usual The results are shown in Fig. 2 and indicate that mice splenectomized 7 days prior to sensitization develop delayed hypersensitivity normally. However, mice splenectomized 21 days before sensitization show a significant (P < 0.05) decrease in their ability to develop delayed hypersensitivity. Splenectomy does not interfere with the ability of antiserum to induce tolerance.

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Doy of Skin lest

Treotmsnts

% Positive

CA J

Anti- Cy serum

0 20 40 60 60

+ t

-,
-

--+ t t42 + t I t ++
FIG. 1. The effects of cyclophosphamide pretreatment on antibody-mediated suppression of delayed hypersensitivity. Two groups of mice were pretreated on Day -2 with 150 mg/kg body wt of cyclophosphamide. On Day 0 one group of mice that received cyclophosphamide was sensitized with 20 pg CA/mouse, and the other was sensitized and suppressed with diluted antiserum having an ABC of 279 gg/mouse. Control groups were sensitized or sensitized and suppressed. Others were left untreated. All mice were then skin tested on Days 14, 28, and 42. Results are from the 28- and 42-day skin tests. Each value represents from 13 to 17 mice. (b) Suppression is significant (P < 0.01) when compared to the sensitized control. (c) Not significantly different (P > 0.1) from mice pretreated with cyclophosphamide and sensitized.

t +

+ +

Transfer of Suppression with Cells and Serum If the inhibition of delayed hypersensitivity induced by antibody is active and due to suppressor cells, as suggestedby the Cy experiment, it should be transferrable with cells from tolerant mice. This was tested using thymus and spleen cells.
Treatments % Positive

0 20 40 60 60
CA w/o CA wlo +As/CA

Shorn splenec day -21 + CA w/o t As/CA Splenec day -7 +CA w/c

FIG. 2. The role of the spleen in antibody-mediated suppression of delayed hypersensitivity. Mice were splenectomized or sham splenectomized on Day -21 or -7. On Day 0 mice were sensitized with 20 pg CA/mouse, or sensitized with CA and suppressed with diluted antiserum to CA having an ABC or 559 pg CA. The mice were then skin tested on Days 14, 28, and 42 as before. Results are from the 28day skin tests. Each value represents from five to nine mice per group. (b) CA w/o vs CA w/o plus As/ CA, P < 0.01; sham-splenectomy Day -7 or -21 plus CA w/o vs sham-splenectomy Day -7 or -21, CA w/o and As/CA, P c 0.01; splenectomy day -7 or -21 plus CA w/o vs splenectomy day -7 or -21, Ca w/o and As/CA, P < 0.01.

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Treatments

Cells or Serum Recipient Transferred Treatments

% Positive

0 20 40 60 80 IO0 CAw/o CAw/otAsKA w/o+As/CA One CRWI0 CAw/otAs/CA CAwio

0 20 40 $0 sb 00 3. The transfer of suppression of delayed hypersensitivity with cells and serum from donors tolerized 7 and 14 days prior to transfer. Mice were: (i) sensitized SCon Day 0 with 0.1 ml w/o containing 20 Fg CA, (ii) sensitized SCon Day 0 with 0.1 ml w/o containing 20 rg CA and tolerized on Days CL 4 with antiserum to CA (ABC = 559 pg), or (iii) inoculated SCwith 0.1 ml w/o containing no antigen and treated with antiserum to CA on Days &4. Seven and fourteen days after sensitization 10 viable spleen cells or thymus cells from these donors were injected iv into naive recipients which were then sensitized SCwith 0.1 ml w/o containing 20 fig CA. Other groups received 0.3 ml serum, ip, from cell donors and then were inoculated SC with CA in w/o. Control animals received 10 spleen cells or thymocytes, or 0.1 ml serum from normal mice. The mice were then skin tested on Days 14, 28 and 42. Results are from the 28-day skin test. Each value represents from 6 to 46 mice. Mice receiving CA in w/o and antiserum were significantly (P < 0.01) suppressed.
FIG.

Figures 3 and 4 summarize the results. Figure 3 shows that IO* spleen or thymus cells from mice tolerized 7 to 14 days earlier, could not transfer inhibition. Threetenths of a milliliter of undiluted serum from tolerized mice also had no effect on sensitization. But Fig. 4 shows that 21 to 28 days after tolerization 10 splenocytes,
Donor Treatments Cells or Serum Recipient Transferred Treatments

CAw/a CAwlo+As/CA w/otAs/CA OW CAW/Q CAw/o+As/CA w/a +AslCA none

CAw/oCAw/o+AslCA CAw/o

CAw/o

ii
CAw/o

c---Ji

FIG. 4. The transfer of suppression of delayed hypersensitivity with cells and serum from donors tolerized 21 and 28 days prior to transfer. Groups and treatments were similar to those in Fig. 3 only cells and serum were transferred 21 and 28 days after sensitization. (b) Significantly different (P-Z 0.05) from mice sensitized only. (c) Significantly different (P < 0.02) from mice sensitized only.

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but not thymocytes, were able to transfer significant (P < 0.05) suppression. Surprisingly, serum from suppressed mice also transferred significant (P < 0.02) suppression. Neither cells nor serum from control groups transferred suppression. Figure 4 also shows that recipients of spleen cells from mice sensitized with CA 21-28 days previously have significantly enhanced (P < 0.05) delayed hypersensitivity reactions when compared to mice only sensitized. These results indicate that the antibody-mediated tolerance investigated here functions by generating suppressor cells and suppressor factors. These cells and serum factors can be detected 21 to 28 days after sensitization and suppression, and require both antigen and antibody for their production. They also suggest that the capacity to transfer an anamnestic response using spleen cells arises 21-28 days after sensitization. Characterization of the Cells Responsible for Transferring Suppression The above experiments suggested that spleen cells from tolerized mice could transfer suppression. We next attempted to identify whether these cells are T or B lymphocytes by treating them before transfer with anti-Ba-b serum or anti-MIg serum, and C. Figure 5 shows that lOa spleen cells could transfer significant suppression when treated with NRS and C (P -K0.02) or anti-BA-6 plus C (P < 0.05). However, after treatment with anti-MIg plus C they could not. Therefore the suppressor cells are B cells. Figure 5 also suggests that T cells capable of transferring an anamnestic response are not present in spleens from suppressed mice. DISCUSSION We have previously described the ability of hyperimmune mouse antiserum to specifically suppress the induction of classical tuberculin-type delayed hypersensitivity in mice (2). However, the mechanisms of this suppression had remained unclear. The present studies have begun to clarify these mechanisms. They show that high doses of Cy, which in other experimental systems eliminated both B and T cells (22, 23), abrogated the tolerance induced by antibody (Fig. 1). This sugcell Treatments Recipient Treatments % Positive

FIG. 5. Characterization of the cells responsible for transferring suppression. Spleen cells from mice tolerized 28 days previously were isolated and incubated with either rabbit anti-BA-0 serum plus guinea pig C, rabbit anti-MIg serum plus C, or normal rabbit serum plus C. Viable cells (10) were injected iv into recipients which were then sensitized with 20 pg CA. Control groups were sensitized with 20 fig CA, sensitized with CA, and suppressed with antiserum to CA (ABC = 559 fig), or left untreated. All groups were skin tested 14, 28, and 42 days after sensitization. Results are from the 28-day skin tests. Each value represents 5 to 10 mice. (b) Significant differences (P < 0.05) exist when compared with the sensitization control.

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gested that suppression is active and cell mediated. However, splenectomy, known to inhibit suppressor T-cell production (13), did not affect tolerance induction. Nevertheless, an active suppressive mechanism seems important because suppression could be transferred with spleen cells (but not with thymocytes) from mice tolerized 21 to 28 days previously (Fig. 4). Interestingly, the suppression was also transferrable with serum from 21- to 28-day tolerant mice. Factors in the serum which transferred suppression most likely were not the originally injected immunoinhibitory antibodies, because 7- and 14-day sera from suppressed animals were ineffective. These should have had more of the originally injected antibody than the effective 21- to 28-day sera. The concomitant appearance of suppressor cells and the serum suppressor factors suggests that the suppressor cells are responsible for the factor production. That the suppressor cells are sensitive to anti-MIg and C, but not anti-BA-8 plus C, and therefore are B cells (Fig. 5), suggests that the transferrable serum factors may be antibodies. Similar to the findings presented here, others also have found B cells to suppress cell-mediated immunologic responses. Katz et al. (24) and Neta and Salvin (25) showed that B cells inhibit skin reactivity in guinea pigs sensitized with purified proteins in Freunds incomplete adjuvant. Zembala et al. (5) were able to inhibit the passive transfer of contact hypersensitivity to picryl chloride using B cells from mice refractory to skin painting. Sy (26) was able to block the passive transfer of immunity to dinitroflurobenzene with B cells from tolerant mice. Sy showed later that suppression of contact hypersensitivity could be transferred with anti-idiotype antibodies, presumably produced by suppressor B cells (26, 27). Binz and his associates (28-30) and Yamamoto et al. (31), have also reported the ability of antiidiotype antibodies to inhibit cell-mediated responses. Several suggestive correlations exist between the humoral suppressor factors described in this paper and anti-idiotype antibodies. Both can suppress celi-mediated responses, and both are produced by injection of immunoglobulins. Antiidiotype antibodies can be induced, and actually were first discovered, using antigen-antibody complexes (32-34). Similarly the production of the suppressive humoral factors requires the presence of both antigen and antibody, indicating that antigen-antibody complexes may be involved in their production. These correlations suggest that the suppressive humoral factors described here may be anti-idiotype antibodies. Production of anti-idiotype antibodies against injected antibodies, which would modulate the degree and duration of delayed hypersensitivity, would be predicted by Jernes network theory (35). Similar regulatory pathways have been demonstrated in other systems (36, 37). However, we have not yet obtained definitive evidence that the humoral factors reported here are anti-idiotype antibodies. The nature and specificity of these factors, as well as the nature and specificity of the B suppressor cells which we postulate are responsible for the production of these factors, is currently under investigation. Preliminary evidence suggests that the humoral factors are antigen specific yet are unable to bind antigen, and that they bear immunoglobulin determinants. The studies reported here and in another paper3 help to identify better what stage of induction is suppressedby antibodies. Crowle discussed previously (1) two characteristics of the long-lived type of delayed hypersensitivity studied here. The first is a more rapid sensitization after a second application of antigen, and the second is that anamnestic responsescan be transferred to syngeneic recipients with

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spleen cells. This secondary type of delayed hypersensitivity has also been described for other antigens (3842), and depends mainly on Thy l+ T lymphocytes (40). The results presented here suggest that spleen cells from mice primed with CA in w/o also can transfer an accelerated response (Fig. 4), and that memory develops 21-28 days after sensitization. Antibody-mediated suppression, though, appears to inhibit the production of T cells capable of transferring anamnesis (Fig. 5) confirming previous evidence by Crowle and Hu (39) that the tolerizing antibodies inhibit early in sensitization. However, other recent results from this laboratory3 show that sensitization, measured by tritiated thymidine incorporation, occurs in antiserum-treated mice by 7 to 14 days. Together with the fact that the tolerizing regimen can be delayed as much as 4 days after sensitization, and still be effective (39), this suggests that the earliest stages of sensitization are not being regulated in this system. Instead, the antibodies appear to inhibit delayed hypersensitivity in later, as yet undefined, steps in sensitization. For example, antibodies may inhibit the production of cells that differentiate into effector and memory cells, or some step in the interactions necessary for generating these cells after initial sensitization has occurred. REFERENCES
1. Crowle, A. J., Advan. Immunol. 20, 197, 1975. 2. Crowle, A. J., and Hu, C. C., J. Immunol. 94, 555, 1965. 3. Crowle, A. J., Yonemasu, K., Hu, C. C., and Fujita, Y., Cell. Immunol. 11, 272, 1974. 4. Yonemasu, K., and Crowle, A. J., Immunology 34, 989, 1978. 5. Zembala, M., Asherson, G. L., Noworolski, J., and Mayhew, B., Cell. Immunol. 25, 266, 1976. 6. Phanuphak, P., Moorhead, J. W., and Claman, H. N., J. Immunol. 113, 1230, 1974. 7. Hendry, W. S., Tilney, N. L., Baldwin, W. M., Graves, M. J., Milford, E., Strom, T, B., and Carpenter, C. B., J. Exp. Med. 149, 1042, 1979. 8. Yamamoto, H., Nonaka, M., and Katz, D. M., J. Exp. Med. 150, 818, 1979. 9. Asherson, G. L., Perera, M. A. C. C., and Thomas, W. R., Immunology 36, 449, 1979. 10. Easmon, C. S. F., and Glynn, A. A., Immunology 33, 767, 1977. 11. Sy, M. S., Miller, S. D., and Claman, H. N., J. Immunol. 119, 240, 1977. 12. Pierce, C. W., and Kapp, J. A., Contemp. Top. Immunobiol. 5, 91, 1976. 13. Sy, M. S., Miller, S. D., Kowach, H. B., and Claman, H. N., J. Immunol. 119, 2095, 1977. 14. Crowle, A. J., and Hu, C. C., Proc. Sot. Exp. Biol. Med. 123, 94, 1966. 15. Arora, M. P., and Crowle, A. J., J. Reticula Endothel. Sot. 24, 271, 1978. 16. Crowle, A. J., Atkins, A., and Hu, C. C., Cell. Immunol. 22, 234, 1976. 17. Golub, E. S., Cell. Immunol. 2, 353, 1971. 18. Reif, A. E., J. Immunol. 89, 849, 1962. 19. Bolton, A. E., Radioiodination Techniques, Review 18. Amersham Corporation, Arlington Heights, Ill., 1977. 20. Hunter, W. M., In Immunochemistry (D. M. Weir Ed.), Vol. 1, pp. 17.1-17.36. Blackwell, Oxford, 1973. 21. Snedecor, G. W., and Cochran, W. G., In Statistical Methods. Iowa State Univ. Press, Ames, 1967. 22. Askenase, P. W., Hayden, B. J., and Gershon, R. K., J. Exp. Med. 141, 697, 1975. 23. Stockman, G. D., Hein, L. R., South, M. A., and Trentin, J. J., J. Immunol. 110, 277, 1973. 24. Katz, S. I., Parker, D., and Turk, J. L., Nature (London) 251, 550, 1974. 25. Neta, R., and Salvin, S. B., J. Immunol. 117, 2014, 1976. 26. Sy, M. S., Cellular Regulation in Contact Sensitivity to DNFB in Mice. Ph.D. Thesis, University of Colorado Medical Center, Denver, 1978. 27. Sy, M. S., Moorhead, J. W., and Claman, H. N., J. Immunol. 123, 2593, 1979. 28. Binz, H., and Wigzell, H., Contemp. Top. Immunobiol. 7, 113, 1977. 29. Binz, H., and Wigzell, H., Nature (London) 262, 294, 1976.

ANTIBODY-MEDIATED 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

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Binz, H., and Wigzell, H., J. Exp. Med. 144, 1438, 1976. Yamamoto, H., Nonaka, M., and Katz, D. H., .I. Exp. Med. 150, 818, 1979. Kelus, A. S., and Gell, P. G. H., J. Exp. Med. 127, 215, 1968. Klaus, G. G. B., Nature (London) 272, 265, 1978. Oudin, M. D., and Michel, M., J. Exp. Med. 130, 595, 1969. Jerne, N. K., Ann Inst. Pasteur Paris 125C, 373, 1974. Bona, C., Hooghe, R., Cazenave, P. A., Leguern, C., and Paul, W. E., J. Exp. Med., 149, 815, 1979. Dohi, Y., and Nisonoff, A., J. Exp. Med. 150, 909, 1979. Crowle, A. J., and Crowle, C. M., J. Zmmunol. 93, 132, 1964. Crowle, A. J., and Hu, C. C., J. Allergy 43, 209, 1969. van der Kwast, T. H., Olthof, J., and Benner, R., Cell. Immunol. 34, 385, 1977. Collins, F. M., and Mackaness, G. B., Cell. Immunol. 1, 253, 1970. North, R. J., and Deissler, F. J., Infect. Immunity 12, 761, 1975.