Role of self carriers in the immune response and tolerance

CELLULAR

IMMUNOLOGY

37, 327-335 (1978)

Role of Self Carriers

in the Immune

Response and Tolerance

III. B Cell Tolerance Induced by Hapten-Modified-Self Active T Cell-Mediated Suppression and Direct DAVID Iv. Divisio?z

of Immmology,

Duke

lJ+ziversity Received

SCOTT

Medical November

Involves Both Blockade l

2

Cmtrr,

Dwham,

North

Carolina,

27710

22,1977

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved irr vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/-l- littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cycle) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclotreated) donors. Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients. In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cycle-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in viva. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors. INTRODUCTION

Haptens coupled to self antigens are usually quite tolerogenic (1-5). Nevertheless, an immune response to hapten-modified self (HMS) can occur under certain circumstances (6-9). Interestingly, similar conditions of hapten-modification were found for the induction of tolerance (5, 8) and both the stimulation of cytotoxic T cells and the labeling of H-2 antigens on the cell surface (6, 8, 10, 11). It was important, therefore, to determine whether cytotoxic T cells or other T cell subclasses played any role in B cell tolerance induction. Our results suggest that B cell unresponsiveness may be regulated by cyclophosphamide-sensitive (T) cells in z&o, as shown in other systems (12, 13), that H-2 labeling is not aZzvu~s necessary, but that B cells may also be directly “blocked” in vitro by hapten-modified self. 1 Supported by American Cancer Society Grant No. IM-89 to D.W.S. and U.S.P.H.S. Grant No. AI-03958 to G. J. V. Nossal. Publication No. 2399 of the Walter and Eliza Hall Institute. 2U.S.P.H.S. Research Career Development Awardee No. AI-00093. The majority of this work was done while the author was an Eleanor Roosevelt Fellow of the International Union Against Cancer, on sabbatical at the Walter and Eliza Hall Institute, Melbourne, Australia. 327 000%8749/78/0372-0327$02.00/O Copyright 0 19;s by Academic E&s, Inc. All rights of repmductfon in any form reserved.

328

DAVID

MATERIALS

W.

SCOTT

AND

METHODS

Animals. Inbred C3H/He X DBA/2 (C3D2), C57BL/6 (both from The Jackson Laboratory, Bar Harbor, Maine), CBA/CaH WEHII, BALB/c nu/nu, and BALB/c nu/+ (Walter and Eliza Hall Institute, Melbourne, Australia) mice of both sexes were used at 6 to 10 weeks of age. The BALB/c nu/nu mice were in their eighth backcross generation when used. They were age and sex matched within each experiment. Preparation of hapten modified self (HMS) I)lfq4plaoid cells. Trinitrophenylated lymphoid cells were prepared as described by Shearer et al. (14) and Scott and Long (8) by incubating IO8 nucleated splenocytes in 1 ml phosphate buffered saline with l-4 ml and 10 mM recystallized trinitrobenzene sulfonic acid (TNBS) for 10 min at 37°C followed by extensive washing. Similar results have been obtained with 0.1-l rnM TNBS, which has been shown to be sufficient to label H-2 and surface immunoglobulin and stimulate cytotoxic effector cells ( 10, 11). Tritiated TNBS and indirect labeling studies revealed that approximately lo7 TNP molecules per cell are covalently associated with the cell surface under these conditions (10, 11, 15, and J. Forman,‘ personal communication). In some experiments, nucleated spleen cells were labeled with fluorescein (FL) by dissolving 4 mg of fluorescein isothiocyanate (BBL, Baltimore, Md.) in 0.05 M carbonate buffer, pH 9.2, and mixing with lo* splenocytes in PBS for 30 min at room temperature prior to extensive washing. Mice were injected in v&o with 2 x 106--lo7 haptenated spleen cells since previous studies demonstrated that as few as lo6 TNP spleen cells were tolerogenic in rats (5). Antigens and in viva iwznzunization. TNP- or FL-ficoll (8) as well as TNP-polymerized flagellin (TNP-POL) were injected interperitoneally into groups of 4 to 5 mice as indicated. Plaque assays were performed at the peak of the direct primary response either by the Jerne method as described earlier (5, 8) or in Cunningham slide chambers ( 16) with TNP-F(ab’) z coated sheep erythrocytes as target cells (17). Earlier results (5) showed that both direct and indirect (IgG) responses are reduced by TNP-SC pretreatment. In this paper only direct PFC are reported for simplicity. Note that “background” PFC were not subtracted from the splenic responses. Hence the percentage control responses in “tolerant” animals would be lower in most experiments. In vi&o culture. Spleen cells were cultured in flat-bottomed Linbro trays (No. FB-TC-16-24) at 5 x lo6 cells/2 ml niicroculture medium (described in Ref. (17) ). Responses were assayed on Days 3 and 4 after initiation of cultures. Teratoma. The- strain 129/J teratoma tissue culture line 402AX (18), which lacks serologically detectable H-2 (19)) was the generous gift of Dr. Linda Gooding, Duke University. Anti-Ly treatment. Spleen cells from the appropriate strains were incubated with anti-Ly reagents and washed before adding thymocyte-absorbed rabbit complement as described earlier (20). Anti-Ly sera of proven specificity were generously provided by Ms. J. Gamble, Dr. J. F. A. P. Miller (Walter and Eliza Hall Institute) and Dr. Ian McKenzie (Austin Hospital, Heidelberg, Victoria). The effectiveness of anti-Ly2 treatment was proven by the demonstration that treated cells failed to generate a cytotoxic response in vitro to TNP-modified syngeneic spleen cells.

SELF

CARRIERS

AND

329

TOLERANCE

RESULTS Kilzetics

of Induction

of Tolerance

by HMS

We have previously reported significant tolerance induction in rats treated with TNP-modified splenocytes (TNP-SC) 1 day before adjuvant challenge (5). Initial efforts to reproduce this phenomenon in mice led to variable results. We, therefore, varied the interval of time between pretreatment with TNP-SC and challenge to determine if a latent period was required for the induction of B cell tolerance by HMS in mice (cf. for example, Ref. 4). The results (Table 1) indicate that tolerance to either TNP or FL in this system requires a latent period of 3 to 4 days, which suggests that an active process may be involved. Failure

to Induce

Tolerance

with HMS

in Nude Mice

We next investigated whether this latent period reflected the activation of T cells for two reasons: 1) numerous tolerance systems have been shown to involve or be

coincident with T suppressor cells (Ref. 4, Zl), and 2) the conditions for tolerance induction

were similar

to those for the stimulation

of cytotoxic

T cells (8, 10, 11).

We therefore wished to determine if nude mice were as susceptible to tolerance induction by HMS as their normal littermate controls. To avoid the transfer of normal T cells in the pretreatment inoculum, we used spngeneic nude spleen cells as “carriers” for TNP. (Preliminary experiments established that nude spleen cells functioned in this capacity) (cf. Ref. (5) ). A s s1lown in Table 2, homozygous nude mice were not rendered tolerant by HMS. This result was observed in three identical experiments. It is noteworthy that the control response in terms of PFC/spleen

of our nude mice to TNP-POL is generally lower than that of their normal littermate (nu/+) controls. This deficit partially reflects the smaller spleen size in our homozygous nude colony since the control data in terms of PFC,/lO” spleen cells are similar

for nu/nu

and nu/+

mice

(not

shown).

To reiterate,

it is clear that

athymic mice are not susceptible to tolerance induction irl T~~VO by haptenated-“self” TABLE

1

Kinetics of Tolerance Induction with TNP-SC or FL-SC? Experiment

1

2

Pretreatment

Interval (days)

Anti-hapten PFC per spleen

Percentage control response

TNP-SC

-7

TNP-SC TNP-SC NSC

-4 -1 -

4,850 8,625 15,933 18,600

f 2,913 f 1,125 f 2,455 f 2,936

26 46 86 100

FL-SC FL-SC FL-SC NSC

-7 -3 -1 -

20,588 18,075 52,412 58,050

f 4,290 f 566 f 9,146 zt 3,858

35 31 90 100 -

a C3D2 mice (five mice per group) injected with lo7 TNP-SC (Expt. l), lo7 FL-SC (Expt. 21, or Normal SC. At various times thereafter, mice were challenged with TNP- or FL-ficoll (10 pg ip) and assayed for anti-hapten PFC on Day 6 after challenge. In Expt. 1, mice were also injectetl with FL-ficoll; this response was not significantly different in all groups and agrees with previously published specificity data from our laboratory (5, 8).

330

DAVID

W.

TABLE Effect

SCOTT

2

of Haptenated Spleen Cells on the ,in I’& of Nude Mice to TNP-POLa

Pretreatment

Anti-TNP

Strain

TNP-nu None

SC

nu/nu nu/nu

TNP-nu None

SC

nu/+ nu- +

Response

PFC/spleen

9,300 f 2,964 (134yc) 6,913 f 1,747 4,063 f 14,233 f

919 ( 28%) 2,534

a Groups of 4 to 5 nu/nu or heterozygous nu/+ littermates were injected iv with 2 X lo6 TNP-nu SC. Four days later, all mice were challenged ip with 20 pg of TNP-POL and 20 rg NIPPOL (the response to NIP was similar in all groups and is not shown for simplicity). Numbers in parenthesis refer to the percentage of the control PFC response made by recipients of TNP-SC on Day 4 after challenge. Similar results were seen in three additional experiments.

spleen cells, a finding which suggests that tolerance in this system is a T-cell dependent phenomenon. Role of Cyclophosphamnide Sensitive T Cells in B Cell Tolerance Induction In the next series of experiments we treated normal BALB/c mice with cyclophosphamide (cycle) at a concentration known to interfere with suppressor T cell activity but which is known to augment cytotoxic and delayed hypersensitivity T cell activity (13, 20). Forty-eight hours later, half of these mice were treated with TNP-SC and all mice challenged with TNP-POL 5 days later. The results are shown in Table 3. Whereas normal mice again are rendered hyporesponsive (38% control response) by HMS, cycle-treated mice were resistant. Similarly (Fig. l), if normal or cycle-treated spleen cells are used to reconstitute nude mice before attempted tolerance induction with TNP-SC, only those mice given normal spleen cells are rendered significantly hyporesponsive compared to their untreated controls. Thus, cycle-sensitive cells seem to be required to induce tolerance to TNP-“self” in this system. This renders unlikely the possibility that cytotoxic or delayed hypersensitivity T cells play a role in B cell tolerance because cyclo-treatment augments these T cell activities (13, 20). TABLE Cyclophosphamide

Interferes

with Tolerance

3 Induction

by Haptenated

Anti-TNP-PFC/Spleen (Day 3)

Spleen Cells

Percentage control response

Cycloa

TNP-SCb

A B

-

+ -

9,800 f 25,200 f

3,380 2,270

38

C D

+ +

+ -

35,800 f 29,500 f

7,380 3,888

121

Group

a 100 mg/kg at -48 hr ip to BALB/c mice (five mice per group). b 107 TNP-nude spleen cells iv. All mice were challenged 4 days later with 20 pg TNP-POL and PFC assayed 3 days later.

ip

SELF

CARRIERS

AND

331

TOLERANCE

w 100 4 2 u-l sow LL d F

60-

i5 v 400 2

CELLS

INJECTED

FIG. 1. Ability of various donor spleen cells to increase the tolerance susceptibility of nude mice. BALB/c nu/nu injected with 3 X 10’ normal SC, 3 X 10’ spleen cells from cyclophosphamide treated donors (100 mg/Kg at - 48 hr) or no cells. Four mice from each group then received 10’ TNP-nude SC iv. All mice were challenged ip with 10 wg TNP-POL and antiTNP-PFC determined 3 days later. Numbers in parenthesis indicate the mean number of PFC per spleen in control mice (receiving no TNP-SC) from two separate identical experiments. Only normal SC reconstitute tolerance susceptibility in nude mice.

In Viva Tolerance by HMS Does Not Require Haptenated H-2 Forman and colleagues have demonstrated that H-Z antigens on the cell surface must be labeled by TNP in order to act as both stimulator and target cells for cytotoxic T cells (10, 11). Similarly, teratoma cells, which lack serologically detectable H-2, fail to act as target or stimulator cells although they can be effectively labeled with TNP (19). It was, therefore, of interest to determine if teratoma cells, labeled with TNP, would induce tolerance in normal recipients. This would indicate whether H-2 needs to be modified with TNP in order to produce an effective “tolerogen” in this system. The results in Table 4 show that TNP-teratoma cells can serve as effective “tolerogens” in viva. TABLE Tolerance

Induction

4

by TNP-Teratoma

Ceils in H-2” Mice

Pretreatmenta

PFC/spleen

A4 B

TNP-teratoma Teratoma

2,930 f 9,929 f

586 2,374

29.5

C D

TNP-SC Normal SC

2,585 f 15,500 f

1,017 3,076

16.6

Group

Percentage control response

a C57BL/6J mice (five mice per group) were injected iv with 10” TNP-labeled (Group A4) or unlabeled (Group B) teratoma cells (from 129/J mice, irradiated with 10,000 r from a r3’Cs source) or with TNP-CS7BL spleen cells (Group C) or with normal spleen cells (Group D). Four days later all mice were challenged with TNPss ficoll (20 pg ip).

332

DAVID

W.

TABLE Effect

5

of TNP-SC (nude) on the in V&W Response to TNP-Polymerized Flagellin of Nude or Ly-2-Depleted Spleen Cells?

Experiment

1

SCOTT

Strain

+TNP-SC”

nu-nu

-

Balb/c

+ + -

+TNP-POti

+ + 2

CBA

CBA

Ly2 + C’

+ + + + -

-

PFC/culture

Percentage control

+ +

11 f lOf6 Sf 312 f 33 f 51 f 551 f 3067 f

2 61 5 11 145 225

+ + + +

10 f 155f 165 f 558 f 142 f 167 f 240 f 1130 f

0 6 56 180 56 35 59 235

+ + -

12

bgd 2.6 bg 17.9 bg 29.5 bg 21.2

0 5 X 106 nu/nu or BALB/c spleen cells were cultured in 2 ml MCM for 4 days before assay vs TNP-SRBC. Response to FL-POL was normal in all groups. b 5 X 105 TNP-nude SC (BALB/c nu/nu in Expt. 1; CBA nu/nu Expt. 2). c 100 rig/ml TNPz. 2 polymerized flagellin (S. adelaide, SW 1338). d Background PFC (no antigen).

Effect of TNP-SC Cultures

on the in Vitro Response of Normal or Ly-2 Depleted Spleen

We and others (8, 22) have previously reported that TNP-SC dramatically reduced the in vitro response of normal spleen cells to the TNP hapten. In order to further analyze the T cell requirements in this process, we repeated these experiments with nude spleen cells and with CBA spleen cells depleted of Ly-2-bearing T cells. The results in Table 5 demonstrate that TNP-SC also “blocked” the in vitro response of nude (Expt. 1) and Ly-2-depleted spleen cells (Expt. 2)) in contrast to the in z&o results (Tables 2 and 3, Fig. 1). Thus T cells do not seem to be required for the in vitro inhibition of the anti-TNP response by HMS. The lower control responses by nude spleen cells in Expt. 1 may reflect that the in vitro response to our TNP-POL is partially T-dependent and does not reflect the toxicity of TNP-SC (unpublished data and Ref. (8) ) . DISCUSSION Haptens coupled to putatively non-immunogenic self carriers have been shown to induce hapten-specific T and B cell unresponsiveness both in z&o and in vitro (l-5, 8, 22, 23). However, haptenated syngeneic spleen cells also can elicit the generation of cytotoxic T cells both in vitro (6, S) and, under certain circumstances, in v&o (13). Since the conditions for the haptenation of spleen cells for the induction of cytotoxicity and tolerance are quite similar (8, 10, ll), we have investigated

SELF CARRIERS

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333

Mhether cytotoxic T cells (or any other T cell populations) are necessary Ior I( cell tolerance induction. The results presented in this paper demonstrate that while T cells seem to be required for ila viva tolerance induction, cytotoxic T cells and DHS T cells are probably not involved. This conclusion is based on the consistent failure to induce tolerance to TNP in nude mice and in cyclophosphamide-treated “normal” mice. While the data for the involvement of T cells are indirect, it should be noted that in preliminary experiments we have found that spleen cells depleted of Ly 2, 3 lymphocytes will augment (2-6 times) the PFC response of nude or cycle-treated mice injected with TNP-SC and TNP-POL. Moreover, the responsiveness of cycle-treated mice to this regimen is reduced by the injection of normal (but not anti-Ly 2 + C-treated) T cells. Finally, we have recently found that anti-8 treatment of “tolerant” spleen cells allows these cells to respond normally to TNP-POL itz vitro (J. Jandinski and D. Scott, in preparation). Since cyclophosphamide treatment has been used to inhibit suppressor cells (13, 24), as well as to nugmcnt the development of DHS and cytotoxicity (20, 13), these results suggest that a regulatory T cell is involved in the induction and maintenance of B cell unresponsiveness in Z$VOand that DHS and cytotoxic cells (while generated in this system) are not directly involved. The nature and exact roles of these regulatory cells are being investigated. The fact that TNP-labeled-teratoma cells can induce “tolerance” (Table 4) but are unable to be recognized by cytotoxic T cells (19), presumably due to their lack of H-2, also suggests that cytotoxic T cells are not mediating this form of U cell tolerance. These data also raise the intriguing possibility that the suppressor cells in this system do not need to “see” haptenated-self H-2. Our results do not mean that haptenated H-2 is not important; rather, we interpret our data to suggest that other haptenated cell surface antigens can be effective tolerogens. It is of interest that haptenated-allogeneic spleen cells can be tolerogenic ( 15, 23, 25). Indeed, in the system used herein (129/J tumor cells into C57BL/6 mice), the tumor cells were H-2 compatible but not syngeneic. However, it has been shown in one system (25) that allogeneic hapten modified cells can induce “phenotypic” tolerance, but the DHS suppressor cells generated in this system only function in H-2 compatible strains. In our B cell tolerance system, the specificity of these regulatory cells is still largely unknown. If they are indeed reactive to modified self, they may be isolatable (26) for further study. In contrast to these in tivo results, we found that both nude and anti-Ly 2 + C treated spleen cells were “tolerized” to TNP by the presence of TNP-SC. This effect may simply reflect the direct inhibition of hapten-reactive B cells by trinitrophenylated moieties (8) shed from the TNP-SC, although the nature of these “tolerogens” and their mechanism of action remain to be determined. We have not as yet titrated the minimal number of TNP-SC (or soluble products from such cells) necessary to inhibit the response of nude spleen cells to TNP-POL. However, in a previous study we found that as few as lo5 TNP-SC profoundly inhibit the ilz vitro responsiveness of lo7 normal splenocytes to TNP-ficoll (S), Since there are approximately lo7 TNP molecules per haptenated cell ( 10, 11, 15)) this corresponds to - 1.6 X 10-l* moles of TNP. This would be equivalent to approximately 25 ng of a tolerogen such as TNPlOHGG. In general, adult (T-dependent) B cells are not rendered tolerant in vitro with 10-100~ higher concentrations of haptenated gamma globulins (27-29). However, T cell independent B cells seem to be more susceptible

334

DAVID

W.

SCOTT

to in vitro tolerance induction (29) ; this may explain the susceptibility of nude or anti-Lyz-treated spleen cells to the in vitro inhibition by TNP-SC. It should be noted that the response to haptenated-POL, in our hands, is only partially T-independent (this paper and Ref. (33) ) and may even vary with the POL batch. Finally, it is curious that anti-Ly, + C treated cells made a small response to TNP-SC alone (Table 5, Expt. 2, line 2). This resembles the in z&o results cited above and is currently being studied. Our data confirm and extend several recent reports that T cells regulate the response to modified self (4, 9, 13, 24, 30, 31). It is worth noting that Miller et al. (4) recently reported both a “direct blockade” and active suppressor mechanism in the control of DHS to reactive haptens, only the latter of which is cycle-sensitive. Despite the reversal of tolerance by anti-6 treatment in vitro (Jandinski and Scott, in preparation), we have not been able to directly demonstrate a dominant suppressor mechanism in our tolerance system in mixing experiments in tivo (Scott, unpublished data). This is not surprising since lack of dominant suppression has been reported in tolerance to haptenated red blood cells (3) even though T cell involvement can be shown (9, 31). Since certain types of suppression can be lost by brief in vitro culture (30) or need to be amplified by antigenic challenge (24), it is possible that the regulatory controls on the B cell resporise to modified self are also subtle and quite labile and may only be required for induction in viva. It is noteworthy that the most clear-cut suppressor systems involving haptenmodified cells are measured by the delayed hypersensitivity responses. Nevertheless, the results presented herein support the conclusion that haptenspecific B cell unresponsiveness can be elicited by HMS via cycle-sensitive cells in viva as well as by direct inhibition in vitro. Whether the same or different selfcarriers are involved in these different pathways remains to be determined. ACKNOWLEDGMENTS I thank Dr. J. F. A. P. Miller for a critical reading of the manuscript; Ms. Jenny Gamble, Drs. J. F. A. P. Miller and I. McKenzie for antisera; Dr. L. Gooding for teratoma cells; and Ms. Louise Klein for performing some of the experiments.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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33.5

18. Gooding, L. R., and Edidin, M., J. Exp. Med. 140, 61, 1974. 19. Forman, J., and Vitetta, E., Proc. Nat. Acad. Sci. (USA) 72, 3661, 1975. 20. 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., 144, 10, 1976. 21. Moller, G. (Ed.), Transplant. Rev. 26, 205, 1975. 22. Naor, D., Mishell, R. I., and Wofsy, L., J. Zmmwtol. 105, 1322, 1970. 23. Miller, S. D., and Claman, H. N., J. Zmmwnol. 117, 1519, 1976. 24. Zembala, M., and Asherson, G. L., Eur. J. Immunol. 4, 799, 1974. 25. Miller, S. D., Sy, M. S., and Claman, H. N., J. Exp. Med. 145, 1071, 1977. 26. Scott, D. W., J. Exp. Med. 144, 69, 1976. 27. Stocker, J. W., Immunology 32, 283, 1977. 28. Cambier, J. C., Kettman, J. R., Vitetta, E. S., and Uhr, J. W., J. Exp. Med. 144, 293, 1976. 29. Cambier, J. C., Vitetta, E. S., Uhr, J. W., and Kettman, J. R., J. Ext. Med. 145, 778, 1977. 30. Starzinki-Powitz, A., Pfizenmaier, K., Rollinghoff, M., and Wagner, H., Ew. J. Zmrrtw~ol. 6, 799, 1976. 31. Naor, D., Saltoun, R., and Falkenberg, F., Eur. J. Immunol. 5, 220, 1975. 32. Scott, D. W., Layton, J. E., and Nossal, G. J. V., J. Exp. Med. 146, 1473, 1977.