IMMUNOLOGICAL UNRESPONSIVENESS IN PRIMED B LYMPHOCYTES

IMMUNOLOGICAL UNRESPONSIVENESS IN PRIMED B LYMPHOCYTES* D. Elliot Parks,? Patricia A. Nelson,$ Sharyn M. Walker,§ and William 0. Weigle Department of Imrnunopathology Scripps Clinic and Research Foundation La Jolla, California 92037

Exposure to antigen can result in either immunological responsiveness or immunological unresponsiveness. The establishment of unresponsiveness in lymphoid cells can be accomplished by a variety of experimental procedures and The nature and can be maintained by any of a number of extent of the unresponsive state is dependent on the form and route of antigen administration as well as the immune status and maturity of the susceptible lymphoid cells. Immature neonatal cells are more readily tolerized than are functionally more mature adult B cells.3-1" However, unresponsiveness has been induced in adult B cells under the appropriate in vitro culture conditions.5, 7 , 11. 14-17 The ability to induce unresponsiveness in individuals primed by previous exposure to antigen would be of limited advantage and has rarely been accomplished.1s-22 The factors responsible for the difficulty in inducing unresponsiveness in primed, as compared to unprimed, lymphoid cells may reflect the maturity of these cells or interference by circulating antibody or activated helper T cells (Th) . Nevertheless, the induction of unresponsiveness in primed B cells has been demonstrated both in vivo 2 3 and in vitro 15-17*24 in a variety of antigenic systems. The mechanisms by which primed B cells can be rendered unresponsive during subsequent antigenic exposure have not been systematically addressed. This paper investigates the induction of immunological unresponsiveness in B cells previously primed by a soluble protein antigen-human gamma globulin (HGG) or the hapten trinitrophenyl (TNP) . Unresponsiveness is established in vivo with the heterologous gamma globulin, HGG, or in vitro with TNP conjugated to turkey gamma globulin (TGG). The kinetics of tolerance induction, the role of suppressor cells, and the surface isotype of the B cells rendered unresponsive are among the parameters assessed in this article.

* This is publication no. 2588 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California. This research was supported, in part, by grants from the United States Public Health Service, nos. A107007 and AG01629, the American Cancer Society, no. IM-42K, and the Biomedical Research Support Program, no. RRO-5514. t Recipient of Junior Faculty Research Award JFRA-8 from the American Cancer Society. t Recipient of a National Science Foundation Graduate Fellowship. This work is submitted in partial fulfillment of the requirements for the Ph.D. degree from the Department of Biology, University of California, San Diego, supported, in part, by a grant from the United States Public Health Service, no. CA09174. § Recipient of Junior Faculty Research Award JFRA-19 from the American Cancer Society. 210 0077-8923/82/0392-0210

$1.75/0 0 1982, NYAS

Parks et al.: Unresponsiveness in Primed B Lymphocytes

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The establishment of tolerance to HGG in A / J mice has been extensively characterized both as to the lymphoid cells rendered unresponsivez5 and the doses of antigen required to tolerize T and B lymphocytes.26.2i The induction of unresponsiveness was attempted in adult A/J mice after priming with 100 pg 1, of immunogenic, heat-aggregated HGG (AHGG) . 2 I As illustrated in FIGURE unresponsiveness could be induced in primed animals by the injection of 2.5 mg monomeric deaggregated HGG (DHGG) 2i if the mice were rested 76 d or more after priming. Although this dose of tolerogen will induce a completely unresponsive state when ifijected into unprimed mice," si injection of tolerogen 10 d following priming with AHGG resulted in the death of the primed mice by anaphylaxis. These results indicate that unresponsiveness can be induced in primed mice, but only if attempted after the level of circulating antibody has diminished. The duration of unresponsiveness established in the spleens of previously primed mice was compared with that of unprimed tolerized mice. Unresponsive-

FIGURE1 , Induction of unresponsiveness following priming.

Days Post Priming

ness is established within 3 d in both T h and B cells of unprimed mice tolerized with 2.5 mg DHGG. Splenic B cells remain unresponsive for at least 45 d following tolerization and Th remain unresponsive for at least three months.27 In contrast, when the same dose of DHGG is injected into mice primed 81 d after priming with AHGG, they are unresponsive for a considerably shorter period of time, as illustrated in FIGURE 2. Although the response of spleen cells from primed mice is depressed as early as 7 d after the injection of tolerogenic DHGG and remains depressed for at least three more weeks, complete responsiveness is observed five weeks after DHGG. These results indicate that both Th and B cells in primed mice recover responsiveness much more rapidly than do the lymphocyte subsets in unprimed tolerized mice. Furthermore, if tested at least five weeks after tolerization, primed tolerized cells demonstrate a response enhanced in comparison to that of primed cells not exposed to DHGG.

Annals New York Academy of Sciences

212

10 I

0

7

I

I

I

It

I

28 Days Post Tolerogen Injection 21

14

35

52

FIGURE 2. Duration of unresponsiveness in primed mice.

The intrinsic ability to induce unresponsiveness in primed spleen cells was investigated by removing those cells from the influence of the primed host. Two to six weeks after priming with AHGG, 60 x lo6 spleen cells were injected into lethally irradiated normal recipients, which were then injected with 2.5 mg tolerogen (DHGG) and subsequently challenged with 400 pg immunogenic AHGG. The results of this experiment are shown in TABLE1, which demonstrates that adoptively transferred normal spleen cells can be rendered unresponsive, whereas transferred primed spleen cells could not be when recipients were tested by challenge 3 d after the injection of tolerogen. However, if antigenic challenge was delayed until 10 d after the transfer and attempted tolerization, unresponsiveness could be established in primed spleen cells (TABLE2 ) . The possibility that the induction of unresponsiveness in primed spleen cells required a longer period of exposure to tolerogen than did induction in normal cells was explored by delaying antigenic challenge of irradiated recipients until 3, 6, 10, or 14 d after reconstitution with primed spleen cells and exposure to tolerogenic DHGG. As shown in FIGURE 3, these experiments demonstrated that unresponsiveness was established in primed and transferred spleen cells but that the unresponsiveness was transient (present only at day 6 to 10 after DHGG treatment). If antigenic challenge was delayed until 14 d after transfer and treatment, primed cells had rccovered responsiveness and were hyperreactive, suggesting that antigen-specific B cells were not functionally deleted by exposure to DHGG but had been unresponsive to challenge on days 6 and 10 due either to receptor blockade or to the presence of suppressor cells. TABLE1 DIFFERENTIAL SUSCEPTIBILITY TO TOLERIZATION IN TRANSFERRED NORMAL AND PRIMEDSPLEEN CELLS

Unresponsive Spleen Cells

DHGG

PFC per 10' Cells

Normal Normal Primed Primed

-

300

+ +

(% )

40 440

87

810

0

Parks et al.: Unresponsiveness in Primed B Lymphocytes

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TABLE2 INDUCTION OF UNRESPONSIVENESS IN PRIMED SPLEEN CELLS AFTER ADOPTIVE TRANSPER Interval Between DHGG and Challenge

Unresponsive PFC per 10' Cells

DHGG -

3 3 10 10

1720 1450

++

N.S.

(% 1

16

550

80 p < 0.05

8.5

In order to assess their ability to induce unresponsiveness in primed B cells, splenic T cells were removed from primed spleen cell populations. Normal and primed spleen cells were treated with rabbit anti-thymocyte serum (ATS) plus C' immediately before adoptive transfer into normal irradiated recipients. As described for the transfer of unseparated primed spleen cells, 2.5 mg DHGG was injected 2 to 4 h after reconstitution with isolated splenic B cells.23 30 X lo6 primed thymocytes were injected as a source of T h Z Rat the time of antigenic challenge, 3 to 14 d after transfer and DHGG treatment. Unresponsiveness could be established in both normal and primed B cells if these cells were transferred and tolerized in the absence of T cells (TABLE3 ) . When challenged 14 d after exposure to tolerogen, transferred isolated primed B cells remained 95% unresponsive (TABLE 3 ) , whereas unfractionated primed spleen cells were several times more responsive than primed cclls not cxposed to DHGG (FIGURE3 ) . In order to determine the kinetic profile of the unresponsiveness induced in primed B cells following transfer and tolerization, the recipients were challenged 3, 6 , 10, or 14 d after reconstitution with Th and antigen. As illustrated 4, unresponsiveness in transferred primed B cells is established in FIGURE within 3 d of the injection of DHGG and remains for at least two weeks after 4) tolerization. The kinetics of unresponsiveness in primed B cells (FIGURE contrast sharply with the kinetics of transient unresponsiveness induced in 3 ) , but are very similar to the unfractionated primed spleen cells (FIGURE kinetics of unresponsiveness in untreated B cells tolerized in normal animals2; The dose requirements for the induction of unresponsivencss in transferred, primed B cells was also investigated. Following ATS treatment and transfer

goo+ 100)

-

Primed Spleen Cells

f

J

p

e

* a

a

FIGURE3. Kinetics of unresponsiveness in primed spleen cells after tolerogen.

50 40

30 20 10

O

3 6 10 14 Days Between Tolerogen and Antigenic Challenge

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TABLE3 INDUCTION OF UNRESPONSIVENESS IN ISOLATED B CELLSAFTER ADOPTIVE TRANSFER Unresponsive B Cells

DHGG

Normal Normal Primed Primed

-

PFC per 10' Cells

(% )

70

++

< 1 p < 0.05 390 20 p < 0.05

99 95

into normal irradiated recipients, 30 x lo6 primed B cells were exposed to increasing doses of DHGG from 100 pg to 2.5 rng. As depicted in FIGURE 5, unresponsiveness was not established in primed B cells following the injection of 100 pg tolerogen. However, a dose of 500 pg DHGG induced significant unresponsiveness and doses of 1 mg or more induced complete unresponsiveness. These data were obtained from recipients challenged 6 d after reconstitution and tolerization. The dose of tolerogen required to induce unresponsiveness in transferred primed B cells (FIGURE 5 ) is only slightly higher than the dose of DHGG previously reported to be necessary for the tolerization of unprimed B cells in the spleens of normal mice.27 A possible involvement of antigenspecific suppressor cells in the establishment of unresponsiveness in primed lymphoid cells was investigated using recently developed in vitro techniques.29 In this assay, 7.5 >( 1 0 6 spleen cells from primed mice or from irradiated recipients reconstituted with primed cells were assayed for their ability to suppress 7.5 X lo6 HGG-specific target cells. When putative effector cells were 6 ) , suppressor cells cocultured for 6 d with responsive target cells (FIGURE could be detected in all primed spleen cell sources regardless of treatment. During the 6 d coculture, suppressor cells were generated from primed spleen cells, from the spleens of irradiated mice reconstituted with primed cells, and from the spleens of reconstituted mice that were also treated with DHGG. The cells present in primed spleen cell populations responsible for the suppression detected in vitro appear to be T cells. Treatment of transferred or untransferred primed spleen cells with ATS plus C' before coculture with HGG-specific target cells completely abolishes the ability to generate suppressor cell activity (FIGURE7). These data indicate that the precursors and/or inducers of suppressor T cells (Ts) detected by this in vitro assay are present in all primed cell populations investigated.

--• Primed 8 Cells

FIGURE4. Kinetics of unresponsiveness in primed B cells after tolerogen. 10

-*-f

0

3

6

---.------. I

10

I

14

Days Between Tolerogen and Antigenic Challenge

Parks et al.: Unresponsiveness in Primed B Lymphocytes

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FIGURE5. Dose requirements for unresponsiveness in primed B cells.

0 01

0.5'

" " 1

-h

25

Dose of Toletogen in mglml

The ability to generate Ts in vitro from transferred primed spleen cells was compared with the ability to induce unresponsiveness in identical cells. Normal irradiated recipients were reconstituted with primed spleen cells and half of these recipients were injected with 2.5 mg DHGG. 2. 6 , 10, or 14 d after transfer, spleen cells were removed from these recipients and cocultured for 6 d with responsive target cells. As illustrated in FIGURE 8, transferred primed spleen cells were equally capable of generating Ts at any of the times tested after transfer with or without tolerogen treatment. The level of suppression generated was equivalent to that detected in normal mice tolerized with DHGG and previously demonstrated to possess Ts both in vivo :In and in v i t r ~ When .~~ compared with the kinetics of unresponsiveness induced in transferred primed 3), the generation of Ts suppressing HGG-specific B cells spleen cells (FIGURE 100

90 80 % 70 60

2

50

ae 40

30 20 10

0 10 20

FIGURE6. Suppressor cells in primed spleen cell populations.

30

:.

40

C 50

'=

60

s 70 80 90 100 Effector Cells

I &Normal Spleen Control

Primed Spleen

Transletred Traniferred Primed Spleen Primed Spleen + DHGG

216

Annals New York Academy of Sciences

in vitro demonstrates no correlation with the transient unresponsiveness induced in primed spleen cells. Confirmation that in vitro induction of Ts in primed spleen cell populations does not correlate with the establishment of unresponsiveness in these primed 4. Whereas primed spleen cells cells is provided by the data presented in TABLE transferred into irradiated recipients respond to antigen challenge unless treated with DHGG, Ts can be generated from these transferred primed spleen cells in vitro regardless of their exposure or lack of exposure to DHGG in the reconstituted recipient. Therefore, the ability to induce mature effector Ts from inducer and/or precursor Ts in primed spleen cell populations appears to be unrelated to the induction of unresponsiveness in primed B cells with HGG.

100

80

FIGURE 7. Anti-thymocyte serum sensitivity of suppression. 20

I T S + C' Elfector Cells

- + - + Transferred Transferred Primed Spleen Primed Spleen +DHGG

1 s Controls

The presence of mature effector Ts in unresponsive primed spleen cell populations was also investigated. In order to circumvent the induction of Ts during in vitro culture, primed spleen cells were added to ongoing target cell cultures before or after transfer into irradiated recipients and exposure to DHGG. The primed cell population containing putative effector Ts were added for the last 2 d of 6 d cultures. FIGURE 9 illustrates the comparison between the results of the 6 d coculture to detect the in vitro generation of Ts and the 2 d coculture to detect the presence of mature effector Ts. Effector Ts cannot be detected in any of the three primed spleen sources regardless of transfer or 6, Ts exposure to tolerogen. However, as previously demonstrated in FIGURE can be generated from all primed cell populations after 6 d of culture in vifro. It can be concluded from these data that, whereas inducer and/or precursor Ts

Parks et al.: Unresponsiveness in Primed B Lymphocytes

217

Transferred Primad Spleen Transferred Primad Splean *OHGG

El

Norrnll Mice +DHGG

:"

FIGURE8. Kinetics of suppression versus time after transfer.

10

20 30-

.-P 40-

5

* ap

506070-

80-

I

2

6

I

10

14

Days Post Transfer and Toleragen

are present in primed spleen cells, the induction or presence of Ts is not responsible for the unresponsiveness that can be induced in primed B cells to HGG. Helper T cells may, however, play a role in the prevention of tolerance induction in primed spleen cells. As illustrated in FIGURES 3 and 4, unresponsiveness is more readily induced and persists for a longer period in isolated B cells than in unfractionated spleen cells. Primed thymocytes or primed splenic T cells 2 n were injected into recipients reconstituted with primed B cells during attempted TABLE 4 In Vivo RESPONSIVENESS Versus in Vitro SUPPRESSION

In Vivo Donor Cells

DHGG

Primed spleen Primed spleen

PFC per 10" Cells

Response

2490 10

100

PFC per Culture

Response

3180 330

100 10

290

9

(% 1