Eur. J. Immunol. 1979. 9: 477-485

Mark D. Grebenau+, David S. Chio and G. Jeanette Thorbecke Department of Pathology, New York University School of Medicine, New York

Tolerance: T cell deletion or suppressor cells ?

T cell tolerance in the chicken 11. Lack of evidence for suppressor cells in tolerant agammaglobulinemicand normal chickens* Tolerance to human y-globulin (HGG) at the T cell level was readily induced in both normal and bursectomized FP and E L 6 strain chickens by intravenous injection of 5 to 50 mg of soluble HGG. T cells from tolerant chickens, subsequently immunized with HGG in complete Freund’s adjuvant (CFA), failed to cooperate in the adoptive immune response to 2,4,6-trinitrophenylated(TNP) HGG with TNP-immune spleen cells. However, suppressive activity could not be demonstrated when cells from tolerant chickens were combined with cells from HGG and CFA-sensitized chickens in the recipients, even at a 5 : 1 ratio. HGG-specific helper activity could be induced, both in bursectomized FP and in intact EL6 spleen cells, but neither tolerant bursectomized nor tolerant intact chicken spleen cells showed suppressor activity in this assay. Such tolerant cells also failed to inhibit formation of helper cell activity upon transfer to intact or bursectomized recipients subsequently exposed to HGG and CFA. Cells mediating delayed hypersensitivity (TDH cells) were also tolerized by intravenous injection of soluble HGG. Injection of cyclophosphamide (CY), 100 mg/kg intraperitoneally, two days prior to the HGG did not interfere with this tolerance induction. CY also failed to augment the D H reactivity of immunized bursectomized animals, although it did increase the reactivity of intact animals. Thus, a CY-sensitive suppressor cell did not appear to be responsible for induction of the tolerant state. Moreover, spleen or thymus cells from tolerant bursectomized or intact animals, transferred into normal chickens, were unable to prevent sensitization of the TDH cells in recipients. This was true in both bursectomized and intact recipients and was also not affected by treatment of recipients with CY, used to facilitate entry of tolerant donor cells into recipients’ lymphoid tissue. Tolerance was also readily induced in thymectomized chickens. It was considered unlikely that the generation of suppressor cells was responsible for induction of tolerance to HGG at the T cell level in bursectomized or intact chickens. Since antibody formation could be excluded as a factor in the bursectomized birds, these restills were in favor of a T cell deletion model of tolerance. Various ways in which delclioii of TDH cells might be achieved are discussed.

1 Introduction Previous work from this laboratory [ l ] has shown that tolerance at the T cell level to the protein antigen human IgG (HGG) can be induced readily in bursectomized, agammaglobulinemic (Ay) as well as in normal chickens. Briefly, it was found that intravenous injection of HGG led to tolerance,

[I 22811

* Supported by Grants AI-3076 and AI-14829 from the United States Public Health Service. +

477

Training Fellow under National Institutes of Health Grant 5T05GM01668. Fellow under United States Public Health Service Training Grant GM00127.

Correspondence: G. Jeanette Thorbecke, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Abbreviations: HGG: Human gamma globulin B A Brucella abortus CFA Complete Freund’s adjuvant TNP 2,4,6,-Trinitrophenyl DH: Delayed hypersensitivity CY: Cyclophosphamide TNBS Trinitrobenzenesulfonic acid SRBC Sheep red blood cells PFC: Plaque-forming cells THceU Helper T cell O E Old tuberculin Ayi: Agammaglobulinemic TDH: T cells responsible for DH Bx: Surgical bursectomy 0 Verlag Chemie, GmbH, D-6940 Weinheim, 1979

whereas presentation with complete Freund’s adjuvant (CFA) was needed to obtain delayed hypersensitivity (DH). The tolerance extended not only to T cells responsible for D H (TDH cells) but also to helper T cells (TH cells). The present experiments were undertaken with the aim of further characterizing the tolerant state. One concept proposed as a mechanism for tolerance induction is that of antigen-antibody complexes blocking the activity of the potential responder cell [Z-51. While this has been used more to explain B than T cell tolerance and may indeed be important at the level of the B cell [ 5 ] ,it certainly cannot be invoked to explain T cell tolerance in the Ay chicken, which produces no antibody of any kind. Moreover, since the kineticsand parameters of T cell tolerance are quite similar for Ay and normal chickens [l], it seems relatively unlikely that two distinct mechanisms would have to be proposed to explain T cell tolerance in the presence and absence of antibody. Regarding the mechanisms which do not involve antibody, a number of possibilities have been proposed. Nossal [6] has suggested a mechanism for low-zone tolerance which involves clonal elimination of a specific population of responder cells through blockage of the T cell receptors. It has also been proposed that low doses of antigen are tolerogenic because of the relative absence of the aggregated component of the antigen 0014-2980/79/0606-0477$02.50/0

478

Eur. J. Immunol. 1979. 9: 477-485

M. D. Grebenau, D. S. Chi and G. J. Thorbecke

needed for proper presentation on macrophages [7]. Phanuphak et al. [8], on the other hand, have postulated that the tolerogen sequesters the responding T cells and takes them out of circulation by inducing them to proliferate rapidly at a site removed from the one where reactivity to the antigen is being studied. These mechanisms share a common basis in that antigen is being presented in the wrong manner or at the wrong site.

at 150000 X g(rmid) for 150 min;theupperthirdofthispreparation was injected into animals within 20 min. The lower two-thirds of the preparation was heat-aggregated at 56 " C for 1 h and used as aggregated antigen. Alum-precipitated HGG was prepared by addition of 5 % KAl(S04)2 to HGG at 12.5 mg/ml [28]. HGG-anti-HGG complex was prepared by mixing the antigen and chicken antibody at 10-fold antigen excess.

Perhaps the most intriguing explanation for tolerance is via the existence of suppressor T cells [9-221 which inhibit the normal functioning of the responder cells either in the efferent [9-111 or aferent [12-201 branches of their activity, directly or via soluble factors [23-241. Antigen-specific suppressor cells have been identified as belonging to certain T cell subpopulations and are much better characterized in the mouse [ll,251 than in other species. In the present study, it will be shown that despite the ease with which chickens can be tolerized to HGG, HGG-specific T suppressor cells cannot be demonstrated at the afferent or the efferent level in either TDH or TH cell systems.

Proskauer and Beck medium cultures of Mycobacterium tuberculosis were grown to stationary phase; the supernatant was used as a source of old tuberculin (OT) (obtained through the courtesy of Dr. I. Millman, Institute for Cancer Research, Philadelphia, PA). It was used at a 1: 10 dilution.

2 Materials and methods 2.1 Animals Strain FP chickens were obtained as eggs from Hy-Line International Production Center, Dallas Center, IA. Line E L 6 chicks were obtained from the Regional Poultry ResearchLaboratory (East Lansing, MI) through the courtesy of Mr. J. Motta. Eggs were incubated and hatched in a Petersime Model 5 incubator-hatcher (Petersime Incubator Co., Gettysburg, OH). Bursectomy was performed by injection of 3.75 mg testosterone propionate (Nutritional Biochemicals Corp., Cleveland, OH) on day 11 of incubation, followed by intraperitoneal injection of 4 mg of cyclophosphamide (CY, Cytoxan, Mead-Johnson, Evansville, IN) on the day of hatching and 3 mg on each of the following days [26]. Ay chickens were used only of their sera failed to give a precipitin line for IgG or IgM when tested with appropriate dilutions of rabbit antichicken Ig antisera which readily detected IgG in a 1: 50 and IgM in a 1 :20 dilution of normal chicken. Ay chickens selected this way never made any antibody to either HGG, Brucella abortus or sheep erythrocytes upon single or repeated injections of antigen. Surgical thymectomy (Tx) was done one day after hatching. Tx animals were autopsied after termination of the experiment to determine whether thymic remnants were present. Newly hatched recipients of cell transfers were routinely treated as follows: 4 mg CY intraperitoneally (i.p.) on day of hatch and 600 R whole body y-irradiation (137Cs-Gammator M, Radiation Machinery Corp., Parsippany, NJ) on the day prior to transfer. Adult transfer recipients were either not treated prior to transfer of cells, or were given i.p. injections of 50 or 100 mg/kg CY 7 days prior to transfer.

A trinitrophenylated preparation of B. abortus was prepared as follows (TNT-BA): 2.0 ml of B. abortus ring test antigen (obtained through the courtesy of Dr. C. E. Watson, US Department of Agriculture, National Animal Diseases Laboratory, Ames, IA) was washed in saline and mixed with an equal volume of 20 mg/ml 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma Chemical Co., St. Louis, MO) in 2% K2C03. This mixture was stirred at room temperature for 2 h, and at 4 " C for 18 h. The conjugated bacteria were then repeatedly washed and centrifuged until the supernatant was free of unconjugated hapten [29, 301. TNP-proteins were prepared as follows: 300 mg of HGG or keyhole limpet hemocyanin (KLH, Mann Laboratories, Orangeburg, NY) dissolved in 7.5 ml of 0.28 M cacodylate buffer, at p H 7.2, was added to 80 mg of TNBS in an equal buffer volume. Following stirring at room temperature for 1 h, the TNP proteins were dialyzed against water to remove unconjugated TNBS. The conjugation ratio was found to be 6-9 mol/105 g protein by spectrophotometry [30,31]. Antigen was administered intravenously (i.v.) or else subcutaneously following emulsification in CFA modified to contain 1 mg of M . tuberculosis (H37 Ra, Difco Laboratories, Detroit, MI) per animal.

2.3 Assays Serum passive hemagglutination titers were determined by titration against sheep red blood cells (SRBC, obtained through the courtesy of the N.Y.C. Department of Health, Otisville, NY), coated with HGG by the CrCI, method [32] or against TNP-SRBC [33]. Sensitized SRBC were tested against antisera of known titers before use. Plaque-forming cells (PFC) were detected by a slide modification of the Jerne plaque assay [34, 351. For PFC development, slides were flooded with a 10% solution of guinea pig complement (absorbed with SRBC) containing rabbit anti-chicken Ig (1 : 1000) and incubated for 45 min at 37 "C.

2.4 Delayed hypersensitivity

2.2 Antigens and immunization procedure HGG was obtained as Fraction I1 from human plasma (E. R. Squibb and Sons, NY).It was passed through DEAE-cellulose using 0.01 M sodium phosphate buffer, p H 8.0 [27]. Deaggregated HGG was prepared by centrifugation of purified H G G

Wattles of chickens were measured with a Schnelltaster automatic caliper prior to and 24 h after injection of antigen into the wattle in a volume of 0.05 to 0.1 ml. On the basis of statistical analysis of results with saline injection, increments in wattle thickness greater than 0.3 mm were considered positive both in male and in female chickens [l],

Tolerance: T cell deletion or suppressor cells ?

Eur. J. Immunol. 1979. 9: 477-485

2.5 Cell transfers Donors were killed by exsanguination, and their spleens were gently teased into 0.165 M Dulbecco’s phosphate-buffered saline. These suspensions were filtered through several layers of gauze, washed, suspended to proper concentration of viable cells, and mixed with antigen (if appropriate) just prior to injection. The vein overlying the tarsus-metatarsus was used in newly hatched recipients; the wing vein was used in adult recipients. Transfer of female cells into male recipients was avoided so as to exclude the possibility of rejection of transferred cells by the recipient because of the sex antigen.

3 Results 3.1 Effect of antigen aggregation on T cell tolerance induction Aggregated forms of protein antigens are regarded as more immunogenic than deaggregated forms, presumably because they are more effectively presented to T and/or B cells by the macrophages [36, 371. While previous data showed clearly that even HGG in incomplete adjuvant invariably induced tolerance at the TDHcell level in Ay chickens, it was still of interest to determine the effect of HGG aggregation on T cell tolerance induction. It can be seen in Table 1 that the i.v. injection of 10 mg HGG, regardless of whether it was deaggregated, alum-precipitated or complexed with chicken anti-HGG, rendered both Ay and normal animals refractory to subsequent sensitization for D H with HGG in CFA. This tolerance was specific since the response to OT was not affected significantly.

479

cells were responsible for this readily obtained absence of DH. In previous studies [1], it was shown that cells from animals sensitized to HGG in CFA acted as helper cells in the adoptive immune response to TNP-HGG when combined with a source of TNP-primed B cells. Cells from animals that had been tolerized prior to sensitization with HGG-CFA failed to provide a helper effect. We therefore tested the effect of mixing cells with known helper activity with spleen cells from tolerized donors. This assay has been used successfully by Basten et al. [9] to demonstrate suppressor cells with HGG specificity in the mouse. The results, summarized in Table 2, show that neither cells from animals receiving HGG i.v. only, nor cells from chickens injected with HGG and CFA after an i.v. injection of HGG caused any detectable inhibition of the helper effect, even when provided in 4-fold excess to helper cells. This failure of tolerant cells to suppress sensitized helper cell activity was observed whether the tolerant cells came from Ay (Expts. 1 and 2) or normal (Expts. 3 and 4) animals. Thymus cells from tolerized donors also failed to suppress helper activity (Exp. 4). It should be noted, however, that cells taken one week after an i.v. injection of HGG, without a further challenge in the donor with HGG and CFA, showed weak helper activity in some experiments (Expts. 2 and 4).

3.2 Attempts to demonstrate suppression of HGG-specific helper cells

In some systems, it has been noted that suppressor cells may be more readily detected as affecting the afferent rather than the efferent branch of the immune response [13, 14, 17-20]. Therefore, we devised an experimental system in which the effect of putative suppressor cells on induction of TH cells might be tested. It was necessary to establish that i.v. preimmunization with HGG and CFA one week prior to immunization with TNP-HGG would significantly increase the response of B cells to TNP in normal animals. This system is shown in Table 3, where a distinct helper effect in animals presensitized with HGG-CFA was observed upon i.v. challenge with TNP-HGG in both the PFC response and also to some extent in the serum anti-TNP levels.

Since aggregated HGG might not be expected to produce tolerance by specific T cell deletion [38] and since inhibition by antibody could not explain the lack of sensitization in Ay animals, it was thought likely that suppressor

Using this experimental design, cells from tolerized donors were injected one day prior to challenge with HGG-CFA, and the recipients were challenged i.v. 7 days later with TNPHGG. Although the results of Exp. 1 (Table 4) showed a sig-

Table 1. Inhibitory effect of i.v. injected H G G on the induction of DH to H G G and OT in Ay and normal chickensa)

I)tI to:’,’

1.,\p.

HGCi injected i.v.

Animal\ (No.)

NO.

I

5- 1 0 mg deaggrcgated 10 mg alum-ppt. None

-

7

1 0 mg alum-ppt. 10 nig HGG anti-HGGd’ None

+

NP

n)

ti(;(; Avg. A wattle Incidence of thickness rerponilcr\ (“) (nlln It Ski.)

o-r

A\g. A wattlc

thickne\\ (nirn 2 S,t-:.)

Nl ( 4 ) NI ( 3 0 )

0.2 f 0.05 0. I f 0.05 1 .o k 0. I 6

17 0 70

I . ( I f 0.10 I . 1 f0.29 1.4 0.27

Ay Ay

(4) (3)

I).I 2 0 . 0 2 I).I f 0 . 0 2

0

Ay

(4)

0.7 f 0 . I4

0.s t 0 . Ih 0.0 ? 0.45 0.7 k 0.18

(1

0 7.5

*

100 100

75

a) Sensitization was performed by subcutaneous injection of 200 pg heat-aggregated H G G in CFA. b) On days 14 and 15 after sensitization, wattle thickness measurements were made before and 24 h after injection of 200 pg H G G and of OT into right and left wattles, respectively. Animals were considered as responders when the wattle thickness increase (A) exceeded 0.3 mm. c) N1 = normal. d) Chicken anti-HGGplus H G G (10 mg) in 10-fold antigen excess.

Eur. J. Immunol. 1979. 9: 477-485

M. D. Grebenau, D. S. Chi and G. J. Thorbecke

480

Table 2. Failure of tolerant spleen cells to suppress sensitized helper cell activity in the adoptive anti-TNP response No. lymphoid cclls x l o - ' injected"' Strain

Ff'

No. of HCX TNPanimal\ "immune" "tolerized" immune

t3p. No.

I

-7

2.6 -c 0.57

2

1 1 I 1

9

0

2

0

3

3

8

0 0

2

2 . 7 f 0.38 6.9t(1.35 3.4 t 0.59 4.3+0.30 5 .f) 0.70 5 . 1 tO.98

7

0

7

3

x

0

7

6

8 0

EL6

3

4 3

5 5 4

Recipient*."' serum titen (log, tSII)

2 2

3 3 0 0 2

4

2 2

6

0

7 6

2

6

2

0

2

2

6.OtO.X3

3.5 t0.17 h.ltO.40

*

0

1 1 1

6

0

s 1.0 2.xIko.92 I.XfO.SX 3.8+ 1.07 s 1.0

0 0 10 10 10"

1

4.5+0.57

I 1 I

0.9Ik0.08

0 0 b

I

1

6.3 -t 0.40 7.3k0.48 5.7 f0 . 5 1

Table 3. Ability of normal chickens to produce T, activity for the antibody response to TNP-HGG after sensitization with H G G in CFA

Response to TNP upon challenge with TNP-HGG i.v.b) PFC/spleen" [n] Serum titers') [nl Erp. No. HGGCFA"' I 3

-

+ +

5365 (1.66)[7]"' 13640(1.51)[X]" 416869(1.74) [4In 1949845 (1.41) [S]"

8.9+1.59[4]

11.3f0.99[S] 12.SIk0.65 [4]

11.3Iko.41 [S]

a) 200 pg H G G in CFA injected subcutaneously on day 0; TNP-BA injected on days -7 and - 14 in Exp. 1 or on day - 14 only in Exp. 2. b) TNP,,, HGG (0.2 mg Exp. 1 or 1 mg Exp. 2) injected i.v. on day 7; PFC assay and serum titrations performed on day 13. PFC per spleen were significantly increased in groups receiving HGG-CFA (p 0.3 mm. d) O.lcp 0.3 mm.

at least 3 weeks longer [l]than one might realistically expect a population of lymphocytes to stay out of the circulation without specific attempts to drain away activated efferent cells from the lymph [58-601.

The ease with which an i.v. injection of HGG induces tolerance at the T cell level in the chicken, even when an aggregated form of HGG is used, suggests that the antigen has to be presented in a special manner in order to stimulate TD, cells, i.e. with an appropriate adjuvant [l,441. In the present study, a low degree of helper activity was initially found in spleen cells from chickens injected with HGG i.v., but after subsequent callenge with HGG in CFA, activity both in TDH and in TH was lacking. Whereas, in the mouse, T D H cells may have a somewhat lower sensitivity to tolerance induction than THcells [48], other studies suggest very similar properties of TDHand T, cells [49, 501. Recent evidence suggests that there may be two synergistically acting TH cells [51, 521, both with similar sensitivity to tolerance induction [53]. Thus, although a family of slightly different T cells may be involved in all these activities, they will be considered together in the following discussion. Since this one family of T cells (in the mouse, Ly-1+,2-,3- [54, 551) is responsible for DH, provides help to B cells and to cytotoxic T cell precursors, as well as factors causing macrophage activation [56], their deletion is pivotal in obtaining operational tolerance to most antigens. Our understanding of natural autotolerance, therefore, to a large extent hinges on the explanation for tolerance at the level of this T cell.

Injection of CY augments the TDHcell response in normal, but not in Ay chickens. It is thus likely that in the present experiments, the drug exerts its effect by removing antibodyforming cells rather than T suppressor cells, unless Ay animals are lacking antigen-specific suppressor cells as well as B cells, as suggested by Droege [61]. If, however, CY were removing such an antigen-specific suppressor T cell, it would be expected to prevent tolerance induction in normal chickens. The data show that CY interferes with tolerance induction neither in intact nor in Ay animals. Although in other laboratories [39-43, 621 antigen-specific suppressor cells, affecting DH reactions or other forms of T cell immunity in mammals, were removed by CY, tolerance induction was prevented by CY administered just prior to the tolerizing antigen injection in some 1401, but not in most of these systems [41, 621. However, recent reports indicate that not all forms of suppressor activities are prevented by prior CY injection [63]. For this reason, we injected CY after the tolerizing antigen dose in some experiments, assuming that suppressor cells might be induced to proliferate by the exposure to antigen and would therefore be especially sensitive to CY at this time. This treatment scheme also failed to prevent tolerance induction. Suppressor cells, postulated to mediate induction of tolerance, are usually found in thymus as well as spleen [57, 641. The observations that direct injection of antigen into thymus [65] or the presence of the thymus facilitates tolerance induction [66, 671 have been interpreted as being due to suppressor cell prevalence in this organ. In addition, the fact that immature thymus is particularly rich in suppressor cells [68] has been used to explain the relative susceptibility to tolerance induction observed in neonates, but a recent study on tolerance to HGG in neonatal mice has failed to provide evidence for antigen-specific suppressor cells in such mice [69]. The suppressor cell that is prominent in neonatal thymus, moreover, suppresses antibody production against both T-dependent and T-independent [68] antigens and is therefore unlikely to have T cell functions as its primary target.

Intravenous injection of the antigen may also be particularly tolerogenic because the spleen is a major source of suppressor cells [57]. Simple sequestration of the antigen and, consequently, of the reactive cells in the spleen cannot readily explain T cell tolerance in the present system since it lasts for

Suppressor cells that depress T cell function such as helper activity [9, 111, DH [16] or contact sensitivity [lo, 141 have been demonstrated in tolerized animals. However, a definite role of these suppressor cells in maintenance of tolerance was discounted by some [48], and a role in induction of tolerance

4 Discussion

484

Eur. J. Immunol. 1979. 9: 4 7 7 4 8 5

M. D. Grebenau, D. S. Chi and G. J. Thorbecke

was considered unlikely by others 19, 69, 701 because demonstration of suppressor cells showed temporal differences with tolerance induction or persistence. Nature of antigen structure requirements [9], dependency on presence of spleen [71], and CY sensitivity [41, 621 also tend to differentiate between suppressor cell and tolerance induction, although similarities between suppressor cell and tolerance induction properties of some antigens are striking [19, 20, 721. On the other hand, failure to demonstrate suppressor cells in adult mice tolerant at the T cell level has also been reported [46, 47, 731. Both the TH cells providing specific and nonspecific helper effects are tolerized by HGG without any evidence for suppressor cells [53]. In the rat, even in a system in which, unlike the present one, dependency on the presence of the thymus for tolerance induction exists, evidence for mediation by suppressor cells has not been obtained (J. Phillips-Quagliata, personal communication). Our experiments indicate, similarly, that HGG-specific suppressor cells may not be present in chickens tolerized to HGG at the T cell level. This is in agreement with results of Hraba et al. [74], who in experiments on T and B cell tolerance induction to bovine serum albumin in newly hatched chickens, have not detected any active suppression. The absence of antigen-specific suppressor cells in the present findings contrasts strongly with results from another system in which spleen cells from Ay chickens cause suppression of antibody production to any antigen, including T-independent responses, and the suppression is clearly directed against B cells [26, 751. Moreover, although in the present study even 50 mg HGG has not led to induction of detectable suppressor cells, in vitro results obtained in this laboratory using SRBC as the antigen (D. S. Chi, M. D. Grebenau and G. J. Thorbecke, manuscript in preparation) show that i.v. injection of SRBC induces antigen-specific suppressor cells in the spleen of chickens within 2 days. Droege [61, 761 and Moticka [77] have suggested that normal chicken thymus cells may cause suppression of antibody production and graft rejection, but in none of these studies have antigen-specific suppressor cells been found in thymus of Ay chickens. This is relevant since Ay chickens in our experiments become tolerized by H G G in lower doses than do intact chickens [l]. An absence of antigen-specific suppressor cells in Ay animals, however, need not mean that a whole class of T cells is absent from the thymus, due somehow (as postulated in 1611) to a deletion of their precursors through bursectomy. It could, for Antigen

-

Immature T cell (thymus1

TH.DHdeletion (terminal differentiation? I (clonal abortion? ) lother mechanism? I

antjgen

Ts proliferation. differentiation ?

TW,DU activation. proliferation, differentiation

"Immunogenic" antigen

Macrophage "activation"

Scheme 1. Possible cellular interactions with antigen leading to deletion or proliteration of T cells responsible for DH and/or helper activity (TH,DH). Note that this scheme suggests induction of suppressor T cell (T,) proliferation on the basis of interaction between antigen, Ts and TH,DH.

example, just as readily be explained on the basis of a failure of suppressor cells to proliferate in the absence of B cells or B cell products, as, for instance, some anti-idiotype suppressor T cells might. The most likely mechanism for production of T cell tolerance in the present studies appears to be that of temporary deletion of specific T cells. Various mechanisms can be imagined for such an effect of tolerogen injection (Scheme 1). One, proposed by Jerne [78], is that immature T cells during proliferation in the thymus are inactivated by untimely exposure to antigens. Thymus dependency of the tolerance induction to HGG has not been demonstrated in the present study, and the rapid rate at which tolerance is established suggests that peripheral T cells are tolerized. It is relevant to note that Chiller et al. [79] found both thymus and peripheral T cells to be tolerant in mice within one day after injection of deaggregated HGG. Another suggested mechanism [7, 801 is that T cells, reacting directly with antigen in the absence of appropriate presentation by macrophages, are somehow deleted rather than activated. A direct confrontation between T cells and antigen in the absence of additional mitosis-stimulating influences might lead to deletion through a form of terminal differentiation. It is also possible that presentation of the antigen to TDH or T, cells via another lymphoid, i.e. T cell (B cells being absent), leads to their inactivation. Such presenting T cells would correspond to the definition of suppressor T cells, although they might not always be induced to proliferate sufficiently to become demonstrable in transfer systems (Scheme 1). Just as Ia is an integral part of the antigen complex required to stimulate TH and TDH cell activation [81], in mice, it may be part of the complex required to induce suppressor T cell proliferation [23, 241. This latter mechanism is particularly attractive in view of the findings of others that antigen (hapten) bound to syngeneic cells, particularly when injected i.v., is very tolerogenic [82]. We greatly appreciate the helpful discussions with Dr. Stephen P. Lerman and the excellent technical assistance of Messrs. Pedro Sanchez, Melvin Bell and Mike Kraft. Received September 10, 1978; in revised form December 18, 1978

5 References 1 Grebenau, M. D. and Thorbecke, G. J., J . Immunol. 1978. 120: 1046. 2 Mackaness, G. B., Lagrange, P. H., Miller, T. E. and Ishibashi, T., J . Exp. Med. 1974. 139: 543. 3 Axelrad, M. and Rowley, D. A., Science 1968.160; 1465. 4 Crowle, A. J. and Hu, C. C., J . Allergy 1968. 43: 209. 5 Diener, E. and Feldmann, M., Transplant. Rev. 1972.8: 76. 6 Nossal, G. J. V., Adv. Cancer Res. 1974. 20: 93. 7 Frei, P. C., Benacerraf, B. and Thorbecke, G. J., Proc. Nat. Acad. Sci. USA 1965.53: 20. 8 Phanuphak, P., Moorhead, J. W. and Claman, H. N., J . Immunol. 1975.114: 1147. 9 Basten, A,, Miller, J. F. A. P. and Johnson, P., Tramplant. Rev. 1975.26: 130. 10 Asherson, G. L. and Zembala, M., Proc. R. Soc. London, Ser. B 1974.187: 329. 11 Zan-Bar, I., Murphy, D. B. and Strober, S., J . Immunol. 1978. 120: 497. 12 Gershon, R. K. andKondo, K., Immunology 1971.21: 903. 13 Doyle, M. V., Parks, D. E. and Weigle, W. O., J . Immunol. 1976. 116: 1640.

Eur. J. Immunol. 1979. 9: 477-485 14 Phanuphak, P., Moorhead, J. W. and Claman, H. N., J. Immunol. 1974. 113: 1230. 15 Ha, T.-Y. and Waksman, B. H., J. Immunol. 1973.110: 1290. 16 Ramshaw, I. A,, Bretscher, P. A. and Parish, C. R., Eur. J. Immunol. 1976. 6: 674. 17 Benjamin, D. C., J . Exp. Med. 1975.141: 635. 18 Weber, G. and Kolsch, E., Eur. J. Immunol. 1973.3: 767. 19 Bullock, W. W., Katz, D. H. and Benacerraf, B., 1. Exp. Med. 1975.142: 261. 20 Bullock, W. W., Katz, D. H. and Benacerraf, B., J. Exp. Med. 1975.142: 275. 21 Nachtigal, D., Zan-Bar, 1. and Feldman, M., Trunsplant. Rev. 1975.26: 87. 22 Fujiwara, M. and Kariyone, A,, Immunology 1978.34: 51. 23 Tada, T., Taniguchi, M. and David, C. S., J. Exp. Med. 1976. 144: 713. 24 Greene, M. I., Pierres, A,, Dorf, M. E. andBenacerraf, B., J. Exp. Med. 1977.146: 293. 25 Cantor, H. and Boyse, E. A,, Transplant. Rev. 1976. 33: 105. 26 Palladino, M. A,, Lerman, S. P. and Thorbecke, G. J., J. Immunol. 1976.116: 1673. 27 Chiller, J. M. and Weigle, W. O., J. Immunol. 1971.106: 1647. 28 Chase, M. W., in Williams, C. A. and Chase, M. W. (Eds.), Methods in Immunology and Immunochemistry, Academic Press, New York 1967, Vol. 1, p. 201. 29 Lerman, S. P., Romano, T. J., Mond, J. J., Heidelberger, M. and Thorbecke, G. J., Cell. Immunol. 1975. 15: 321. 30 Little, J. R. and Eisen, H. N., in Williams, C. A. and Chase, M. W. (Eds.), Methods in Immunology and Immunochemistry, Academic Press, New York 1967, vol. 1,p. 128. 31 Rittenberg, M. B. and Amkraut, A. A,, J. Immunol. 1966. 97: 421. 32 Poston, R. N., J . Immunol. Methods 1974.5: 9 1. 33 Rittenberg, M. B. and Pratt, K. L. Proc. SOC. Exp. Biol. Med. 1969. 132: 575. 34 Jerne, N. K. and Nordin, A. A,, Science 1963.140:405. 35 Mishell, R. I. andDutton, R. W., J . Exp. Med. 1967.126.423. 36 Unanue, E. R. and Askonas, B. A., Immunology 1968. 15: 287. 37 Mitchison, N. A., Immunology 1969. 16: 1. 38 Dresser, D. W. and Mitchison, N. A,, Adv. Immunol. 1968.8: 129. 39 Askenase, P. W., Hayden, B. J. and Gershon, R. K., 1. Exp. Med. 1975.141: 697. 40 Polak, L. and Turk, J. L., Nature 1974.249: 654. 41 Sy, M.-S., Miller, S. D. and Claman, H. N., J . Immunol. 1977. I 1 9: 240. 42 Bash, J. A,, Singer, A. M. and Waksman, B. H., J . Immunol. 1976. 116: 1350. 43 Rollinghoff, M., Starzinski-Powitz, A., Pfizenmaier, K. and Wagner, H., J . Exp. Med. 1977.145:455. 44 Palladino, M. A,, Grebenau, M. D. and Thorbecke, G. J., Dev. Comp. Immunol. 1978.2: 121. 45 Kolsch, E., Stumpf, R. and Weber, G., Tranrplant. Rev. 1975. 26.57. 46 Zolla, S. and Naor, D., J. Exp. Med. 1974. 140: 1421. 47 Scott, D. W., J. Immunol. 1973. 111: 789. 48 Silver, J. and Benacerraf, B., J. Immunol. 1974. 113: 1872.

Tolerance: Tcell deletion or suppressor cells ?

485

49 Kettmann, J., Immunol. Commun. 1972.1: 289. 50 Kerckhaert, J. A. M. and Hofhuis, F. M. A,, Ann. Immunol. Pans 1975.126C: 26. 51 Tada, T., Takemori, T., Okurnura, K., Nonaka, M. andTokuhisa, J. Exp. Med. 1978. 147: 446. 52 Janeway, C. A., Murgita, R. A., Weinbaum, F. I., Asofsky, R. and Wigzell, H., Proc. Nut. Acad. Sci. U S A 1977. 74: 4582. 53 Hoffeld, J. T., Marrack, P. and Kappler, J. W., J . Immunol. 1976. 117: 1960. 54 Vadas, M. A,, Miller, J. F. A. P., McKenzie, I. F. C., Chism, S. E., Shen, F.-W., Boyse, E. A., Gamble, J. R. and Whitelaw, A. M., J. Exp. Med. 1976. 144: 10. 55 Pang, T., McKenzie, I. F. C. andBlanden, R. V., Cell. Immunol. 1976. 26: 153. 56 North, R. J., Cell. Immunol. 1973. 7: 166. 57 Gershon, R. K., Lance, E. M. and Kondo, K., J . Immunol. 1974. 112: 546. 58 Atkins, R. C. and Ford, W. L., J. Exp. Med. 1975.141: 664. 59 Hay, J. B., Cahill, R. N. P. and Trnka, Z., Cell. Immunol. 1974. 10: 145. 60 McConnell, I., Lachmann, P. J. and Hobart, M. J., Nature 1974. 250: 113. 61 Droege, W., Eur. J. Imrnunol. 1973.3:804. 62 Clark, C., Azar, M. M. and Gleason, D. F., Cell. Immunol. 1976. 26: 228. 63 Zembala, M. and Asherson, G. L., Clin. Exp. Immunol. 1976. 23: 554. 64 Ha, T.-Y. and Waksman, B. H., J. Immunol. 1973.110: 1290. 65 Horiuchi, A. and Waksman, B. H., J. Immunol. 1968.101: 1322. 66 Gershon, R. K. and Kondo, K., Immunology 1970.18: 723. 67 Phillips-Quagliata, J. M., Bensinger, D. 0. and Quagliata, F., J . Immunol. 1973.111: 1712. 68 Mosier, D. E., Mathieson, B. J. and Campbell, P. S., J . Exp. Med. 1977. 146: 59. 69 Benjamin, D. C., J. Immunol. 1977.119:311. 70 Askenase, P. W., Hayden, B. and Gershon, R. K., J . Immunol. 1977.119: 1830. 71 Sy, M.-S., Miller, S. D., Kowach, H. B. and Claman, H. N., J. Immunol. 1977.119: 2095. 72 Thkze, J., Waltenbaugh, C. and Benacerraf, B., Eur. J . Immunol. 1977. 7: 86. 73 Dwyer, J. M. and Kantor, F. S., J . Exp. Med. 1975.142: 588. 74 Hraba, T., Karakoz, I. and Madar, J., Folia Biol. Prague 1977. 23: 336. 75 Grebenau, M. D., Lerman, S. P., Palladino, M. A. and Thorbecke, G. J., Nature 1976. 260:46. 76 Droege, W., Proc. Nut. Acad. Sci. USA 1975. 72: 2371. 77 Moticka, E. J., J. Immunol. 1977. 119: 987. 78 Jerne, N. K., Eur. J . Immunol. 1971. I : 1. 79 Chiller, J. M., Louis, J. A,, Skidmore, B. J. and Weigle, W. O., in Katz, D. H. and Benacerraf, B. (Eds.), Immunological Tolerance, Academic Press, New York 1974, p. 373. 80 Katz, D. H. and Unanue, E. R., J . Exp. Med. 1973.137:967. 81 Shevach, E. M., J. Immunol. 1976. 116: 1482. 82 Claman, H. N. and Miller, S. D., J. Immunol. 1976. 117: 480.