Extrathymic tolerance of mature T cells: Clonal elimination as a consequence of immunity

Cell, Vol. 63, 1249-1256,

December

21, 1990, Copyright

0

1990 by Cell Press

Extrathymic Tolerance of Mature T Cells: Clonal Elimination as a Consequence of Immunity Susan Webb, Claudia Morris, and Jonathan Department of Immunology Research Institute of Scripps Clinic 10666 North Torrey Pines Road La Jolla, California 92037

Sprent

Summary The mechanism by which T lymphocytes an? tolerized to self or foreign antigens is still controversial. Clonal deletion is the major mechanism of tolerance for immature thymocytes; for mature T cells, tolerance is considered to reflect anergy rather than deletion, and to be a consequence of defective presentation of antigen. This paper documents a novel form of tolerance resulting when mature T cells encounter antigen in immunogenic form. Evidence is presented that exposure of mature T cells to Mlsa antigens in vivo leads to specific tolerance and disappearance of Mlsa-reactive 436’ T cells. Surprisingly, the clonal elimination of VpS+ cells is preceded by marked expansion of these cells. Thus, tolerance induction can be the end result of a powerful immune response. These data raise important questions concerning the relationship of tolerance and memory. Introduction The issue of how T lymphocytes respond to a wide variety of foreign antigens while avoiding autoaggression has been a topic of intensive investigation for many years (reviewed in Schwartz, 1989). Although T cells are generally unresponsive to self components, the mechanism of self tolerance induction is still poorly understood. Nevertheless, recent work has provided direct evidence that T cell tolerance can reflect at least two different mechanisms: clonal deletion (Kappler et al., 1987, 1988; MacDonald et al., 1988b; Kisielow et al., 1988; Sha et al., 1988; Fowlkes et al., 1988) and anergy (Lo et al., 1988; Qin et al., 1989). In the case of immature T cells, it is now clear that intrathymic exposure to certain cell surface antigens, including major histocompatibility complex (MHC) molecules and minor lymphocyte stimulating (MIS) molecules, can cause T cell deletion at an early stage of differentiation. Clonal deletion appears to be the main mechanism of tolerance when antigen is encountered intrathymically on bonemarrow (BM)-derived cells. For antigen expressed on other cell types, e.g., thymic epithelium, clonal anergy has been invoked: specifically reactive T cells appear not to be deleted but are refractory to subsequent exposure to antigen (Ramsdell et al., 1989; Roberts et al. 1990). In contrast to immature T cells, exposure of mature T cells to antigen generally leads to immunity rather than tolerance. Nevertheless, there is increasing evidence that under some conditions mature T cells do show susceptibility to tolerance. Such tolerance seems to reflect either

suppression or anergy (reviewed in Schwartz, 1989). In the case of anergy, tolerance appears to be a consequence of T cells confronting antigen on inappropriate antigen-presenting cells (APCs) or on damaged APCs (Markmann et al., 1988; Mueller et al., 1989). Significantly, in marked contrast to immature T cells, there is no clear evidence that tolerance of mature T cells reflects clonal deletion (Blackman et al., 1990b). Much of our current understanding of tolerance induction has emerged from studies on MIS molecules. These molecules show limited polymorphism and only two stimulatory forms, Ml@ and MlsC, have been described (Abe and Hodes, 1989); some mouse strains fail to express stimulatory MIS molecules and are typed as Mlsb (Festenstein et al., 1973). Recognition of MIS molecules may be limited to T cells, as attempts to raise antibodies to these antigens have been unsuccessful. MIS antigens are particularly useful tools for studying tolerance for two reasons. First, primary T cell proliferative responses to MIS antigens are extremely high; in fact, they are as high as or higher than responses to MHC antigens (Janeway et al., 1980; Miller and Stutman, 1982). Second, reactivity to MIS antigens is restricted to T cells expressing particular T cell receptor (TCR) Vp elements, e.g., VP6 and VpS.1 for anti-MI+ responses and Vf33 for anti-MIsC responses. These T cells are deleted in Mls-positive mouse strains; thus, Ml@ mice show selective deletion of VpS+ and Vf38.1+ T cells, whereas MlsC mice delete V&Y T cells (Abe et al., 1988; Fry and Matis, 1988; Kappler et al., 1988; MacDonald et al., 1988b; Pullen et al., 1988). Tolerance to MIS antigens can be induced experimentally by transferring Ml+ or Ml+bearing cells into neonatal Mlsb mice (MacPhail et al., 1985; MacDonald et al., 1988a; Waite and Sunshine, 1988). Tolerance in this model is long-lasting and reflects clonal deletion in the thymus. An interesting feature of neonatal tolerance to MIS antigens is that tolerance is elicited principally by antigen expressed on T cells (Webb and Sprent, 1990). Tolerance induced by T cells is largely controlled by CD8+ cells: cell for cell, CD8+ cells are 50- to lOO-fold more potent than other cell types, including B cells. By contrast, immunogenic expression of MIS antigens is detectable only on B cells; thus, stimulation of Mls-reactive T cells or hybridomas can be elicited by purified B cells but not by T cells or typical APC (von Boehmer and Sprent, 1974; Ahmed et al., 1977; Webb et al., 1989). This paper examines tolerance of mature peripheral T cells to MIS antigens. In confirmation of the original report of Jacobsson et al. (1975) recent studies by Rammensee et al. (1989) have shown that transfer of unseparated Ml@ spleen cells to adult Mlsb mice induces specific unresponsiveness to MIS antigens. Tolerance was reported to reflect anergy rather than clonal deletion, strengthening the view that clonal deletion operates only at the level of immature T cells; anergy of Mlsareactive mature T cells has also been observed in chimeras (Ramsdell et al., 1989; Roberts et al., 1990) and in TCR transgenic mice

Cdl 1250

Table

1. Injection

BlO.BR

of Ml@ AKRN

Mice Injected

with:

Not injected AKFVJ (Mb?) CD4+ T AKRN (MW) CD6+ T AKRlJ (Mlsa) T- spleen CBAlCa (Mtsb) CD6+ T

Cells into ATx BlO.BR % of CD4+ LN T Cells Expressing V66 11.1 7.2 3.5 4.5 11.3

Mlsb Mice Induces MLR (cpm BlO.BR 2.6 1.6 1.2 2.3 1.7

(9.1)8 (5.5) (5.3) (10.6) (8.1)

Specific

Tolerance

x 103) by Host CD4+

(MIs~,H-~~)

AKRlJ 126.5 109.7 17.4 44.0 151.6

to Mlsa Antigens Cells Responding (Mlsa,H-2k) (132.2) (100.3) (20.8) (49.9) (163.3)

to: BIO.P

(Ml@,H-2P)

91.9 61.5 50.6 76.6 79.5

Thymectomized BlO.BR mice were injected i.v. with 1 x 10’ highly purified CD4+ or CD6+ T cells or T-depleted (B-enriched)‘spleen cells from AKRlJ or CBAlCa mice. Fourteen days later, CD4+ host (Thy-l .l-) T cells were purified from pooled lymph nodes and tested in MLR for reactivity to either MW or allo-H-2P-bearing T- spleen stimulators. 3H-thymidine incorporation was measured on day 4 in cultures containing 1.5 x lo5 responder cells and 5 x lo5 stimulator cells. The responses shown are the mean of triplicate cultures. a The numbers in parentheses show the response when the cultures were supplemented with 5 U/ml recombinant IL-Z.

(Blackman et al., 199Oa). In the studies reported here we have reevaluated the mechanism of MIS tolerance in adult mice, using thymectomized (ATx) hosts to prevent de novo differentiation of T cells. The results are unexpected in two respects. First, transfer of Ml@ lymphoid cells, including purified CD8+ cells, to Mlsb hosts leads to clonal elimination of the majority of Vj36+ CD4+ T cells. Second, the disappearance of MI.+reactive Vj36+ cells is preceded by a marked expansion of these cells. The main conclusion from these findings is that clonal deletion as a mechanism of tolerance can operate at the level of mature T cells. Susceptibility to clonal deletion is thus not restricted solely to immature T cells. Elimination of mature T cells appears to be the end result of a powerful immune response. Results Induction of Functional MW Tolerance and Deletion of VpS+ T Cells in Adult Mlsb Mice Given MW Lymphoid Cells As discussed above, other workers have reported that injection of Mlsb mice with unseparated Ml@ spleen cells induces specific unresponsiveness associated with clonal anergy: T cells from the host mice respond poorly to Mlsa cells in vitro but show little or no reduction in Vps cells (Rammensee et al., 1989). In preliminary experiments with normal (nonthymectomized) hosts, we confirmed that the MIS* tolerance induced in adult mice leads to unresponsiveness as measured by primary mixed lymphocyte responses (MLRs) in vitro (data not shown). The extent of tolerance was variable, however, and tolerance waned after several weeks. Interestingly, the level of Vj36+ T cells in the injected mice showed considerable variation from one experiment to another. Some mice were partly depleted of VpS+ cells whereas other mice showed no depletion. In subsequent experiments we used adult thymectomized (Alk) mice as hosts to avoid the problem of new T cells (including Vj36+ cells) emerging from the thymus during the period of the experiment. All of the experiments discussed below involved young (6-8 week) adult mice that were thymectomized 4-10 days prior to tolerance induction. To induce tolerance, ATx Mlsb mice were injected intravenously (i.v.) with highly-purified sub-

sets of lymphoid cells prepared from HQcompatible Ml@ mice. In most experiments, BlO.BR (Mlsb, H-2k) mice were injected with AKRlJ (Mlsa, H-2”) or (BlO.BR x AKR/J)F, cells; in some experiments, BlO.D2 (Mlsb, H-2d) mice received DBAR (MIP, H-2d) cells. Tolerance was examined at 2-6 weeks after transfer. Unless stated otherwise, the data in the tables and figures were obtained from cells pooled from two mice per group. In the experiment shown in Table 1, Mlsb mice (BlO.BR) were injected with MIS* (AKRIJ) CD8+ or CD4+ lymph node (LN) T cells or with T cell-depleted (T-) spleen cells. Two weeks later, LN cells from the injected mice were depleted of residual donor (Thy-1.1) T cells and fractionated into CD4+ cells for use as responder cells in MLR. With injection of a dose of 10’ MIS* lymphoid cells, CD8+ cells elicited strong tolerance, the response of host CD4+ cells to Mlsa (AKR/J) in culture being reduced by 385%; addition of interleukin 2 (IL-2) during culture caused little or no improvement in the response (Table 1, data in parentheses). Tolerance was less marked with injection of Tspleen cells and minimal with transfer of CD4+ cells. Tolerance was specific since responses to third-party H-2 (H-2p) antigens showed little or no reduction (see Table 2). Since the donor and hosts differed at various minor histocompatibility (H) loci, control BlO.BR mice were injected with MIS (Mlsb)-compatible minor H-different CBA/ Ca CD8+ cells. These cells failed to elicit tolerance to Mlsa antigens (Table 1). Cell mixing experiments indicated that Mlsa tolerance was not associated with detectable suppression. Thus, addition of tolerant T cells to culture failed to inhibit the anti-Mlsa MLR by normal Mlsb T cells (data not shown). In other studies, marked and specific Ml@ tolerance was also observed in the combination of DBAR (Mlsa) - BlO.D2 (Mlsb) (data not shown). A titration of the dose of Ml@ lymphoid cells required to induce tolerance in Mlsb mice in the AKR/J + BlO.BR combination is shown in Table 2; in addition to the cell types discussed above, the host mice in this experiment were injected with highly purified small B cells (prepared from spleen). CD8+ T cells and purified B cells showed similar potency in terms of tolerance induction. For these cells, doses as low as 1.5 x lo6 reduced the host response to Ml@ antigens (AKRIJ) by 280% (boxed numbers). T- spleen cells were less potent than purified small

Tolerance 1251

of Mature

T Cells

Reflecting

Table 2. Relative Effectiveness Induction of Mlsa Tolerance Number

of

Clonal

of Different

Deletion

Cell Types

Uninjected Controls

in

MLR (cpm x 103) by Host CD4+ Cells Responding to:

Cells Injected (x 106)

BlO.BR (Ml@,H-2’0

AKRIJ (MIs~,H-~~)

BIO.P (Ml+‘,H-2P)

Uninjected

-

2.3

113.2

54.1

AKRlJ

lg+ B

15 1.5 0.15

3.4 2.4 3.4

022.8 22.9 98.9

53.6 79.3 94.7

AKRN

T- spleen

15 1.5 0.15

4.3 4.0 3.5

123.21 39.6 97.2

62.5 73.4 68.6

AKRlJ

CD4+ T

15 1.5

3.5 2.4

42.6 95.9

86.7 61 .O

AKRIJ

CD8+ T

15 1.5 0.15

4.4 3.6 3.5

0 14.0 15.2 54.3

91.0 73.7 74.4

BlO.BR Injected

Mice with:

Various doses of purified AKRlJ cell populations (see Experimental Procedures) were injected into thymectomized BlO.BR mice. Host CD4+ cells were purified 15 days later and tested in MLR. MLR were measured on day 4. Responses showing 880% specific unresponsiveness to Ml@ are boxed.

B cells, which indicates that large B cells and typical APC such as macrophages and dendritic cells do not play an essential role in tolerance induction; these cells are enriched in T- spleen, but are rare in purified B cell populations. CD4+ T cells were poorly tolerogenic, and even high doses of these cells (15 x 106) caused only marginal tolerance. To examine whether tolerance involved clonal deletion of MI+reactive T cells, LN CD4+ cells from the tolerant Table 3. Tolerance in Mlsb Mice Given MW Lymphoid Cells Associated with Clonal Elimination of Vj36+ Host CD4+ Cells

BlO.BR Injected

Mice with:

Source T Cells

Injected with ma COB+ cdk

of

Is

Percent of CD4+ Cells Expressing:

Percent of CDB+Cells Expressing:

‘JP6 W

‘43’3 ‘438

Uninjected (BlO.BR x AKR/J)F, CD4+ T (BlO.BR x AKR/J)F, CD8+ T (BlO.BR x AKRN)F, T- Spleen

LN LN

9.3 8.1

16.7 16.3

16.8 12.0

28.0 23.6

LN

2.1

15.7

15.5

24.0

LN

2.4

15.1

NT

NT

Uninjected (BlO.BR x AKR/J)F, CD8+ T (BlO.BR x AKR/J)F, T- Spleen

Spleen Spleen

9.0 3.6

16.2 16.5

11.6 11.5

17.9 21.3

Spleen

3.1

16.0

16.9

24.7

1.5 x 10’ (BlO.BR x AKR/J)F, (MW) cells were injected into ATx BlO.BR (Mlsb) mice. LN cells or nylon-wool-passed (T-enriched) spleen cells were prepared on day 17 after injection. Using two-color flow cytometry analysis the percentage of T cell subsets expressing V56 versus V88 (8.1 + 8.2) was determined. NT, not tested.

CD4

CD4

Figure 1. Selective Depletion of VfW CD4+ Cells in Ml.+Tolerant Mice Revealed by Two-Color Flow Cytometry Thy-l.lLN cells from ATx BlO.BR (Mlsb) mice given 1 x 10’ AKFUJ (Ml.sa) CD8+ cells 14 days before were double-stained with anti-V66 (RR47) or anti-V88 (KJ18) MAb in addition to anti-CD4 (GK1.5) MAb (see Experimental Procedures); LN cells from uninjected BlO.BR mice were used as a control. The data are plotted on a log scale. The percentage of total LN cells falling into each quadrant is indicated. The percentages of T (Thy-l+) cells in the injected vs. control mice were 52% and 54%, respectively.

mice were assayed for Vj36 expression. As shown in Tables 1 and 3, mice injected with Ml@ CD6+ T cells or Tspleen cells showed a substantial (SO%-60%) reduction in the level of V66+ cells relative to uninjected control mice. There was little or no decrease in the proportion of VpS+ cells. The reduction of VpS+ cells was apparent in both spleen and LN and was evident only at the level of CD4+ cells (Table 3, Figure 1). The possibility that the reduced level of VpS+ cells reflected TCR modulation is unlikely because the intensity of staining of the residual Vp6+ cells appeared normal and there was a clear distinction between positive and negative cells (Figure 1). The decrease in V66+ cells could not be attributed to the relative expansion of other T cells, since the total numbers of T cells in the tolerant mice were not elevated. The tolerance in Mlsb mice given Mlsa lymphoid cells required the injection of viable cells. Injection of cells exposed to radiation (3000 rad) shortly before injection failed to cause either VP6 deletion (Table 4) or functional Ml@ tolerance (data not shown). With regard to the duration of tolerance, tolerance measured at 6 weeks after transfer (the latest time examined) was as marked as at 2-3 weeks. Clonal Elimination of Vp6+ T Cells in MWTolerant Mice Is Preceded by Marked Clonal Expansion In all of the above experiments, tolerance and Vf36 expres-

Cdl 1252

sion were examined at 82 weeks after lymphoid cell transfer. The clonal elimination of VpS+ cells seen at this time was highly surprising, since some of the cells used for tolerance induction, notably B cells, are known to be strongly immunogenic for Vf36+ T cells, at least in vitro. For this reason it was of interest to determine the tempo of VfW cell deletion. In the two experiments shown in Figure 2, groups of Mlsb mice were given Ml@ CD8+ or T- spleen cells and then killed at various intervals to examine VpS expression. The unexpected finding was that, on day 4 after transfer, levels of VfW cells were markedly elevated. At this stage, up to 40% of the host splenic C&l+ cells were VfW (compared with about 10% in uninjetted control mice). The proportion of host Vf36’ cells fell sharply between day 4 and day 7 and then declined progressively to reach 20/o-3% by day 22. The sharp elevation of Vf36+ cells seen at day 4 after injection was more prominent in spleen than LN, and was associated with little or no change in the frequency of Vf38+ cells. The rise in host VpS+ cells required injection of comparatively few Ml.9 cells. Injection of 2 x lo6 CD8+

Table 4. Irradiation of Donor Ml@ Cells Prevents Elimination of Host Vf36’ Cells in Ml&’ Mice during the Stage of Tolerance Induction

BlO.BR Injected

Experiment 1.

2.

Mice with:

Percentage CD4+ Cells Expressing:

of

436

‘JW

Uninjected AKRIJ Tspleen Irradiated AKWJ T- spleen

9.6 4.6

15.4 14.9

9.1

15.5

Uninjected AKRN CD8+ T Irradiated AKRIJ CD8+

9.8 3.3 10.3

15.4 16.5 14.8

Thymectomized BlO.BR mice were injected with 1 x 10’ AKR/J cells. Some of the mice received cells that were irradiated (3000 rad) just prior to injection. Fourteen days later the percentage of Vf3Gt and V68’ LN CD4+ cells was assessed by fluorescence-activated cell sorter analysis.

Figure 2. The Depletion of Host Vf36+ Cells in Ml& Mice Injected with MIS* Lymphoid Cells is Preceded by Enrichment of V86+ Cells

5

Day

4

I

1

2x10' 1x10

(A) ATx 610. BR (f&b) mice were injected with either 2 x 10s or 1 x 10’ AKR/J (Mlsa) CD8+ cells iv. At the days indicated, LN cells (open columns) or spleen cells (solid columns) were assessed for percentage of CD4+ cells expressing Vf36 TCR. (B) As for (A) except that the host mice received either 1 x 10’ T- spleen cells or 1 x 10’ CD8+ cells from (BlO.BR x AKR/J)F, (MIS*) mice. Counts of total CD4+ T cells recovered from spleen or LN cells of the injected mice showed little or no elevation compared with the uninjetted controls, even at day 4 after injection.

2x10'

.-

1x10'

t; .L

= k

Day

7

0 LN

n Spleen

% V86*

Day

CD4’

T Cells

7 El LN n Spleen

Day 14

10 % V86’

15 CD4’

20 T Cells

25

30

Tolerance 1253

of Mature

T Cells

Reflecting

Day

4 After

Clonal

Deletion

Injection

1 t

I3 1

80

Table 5. Specific Enrichment of V66+ Cells in Mlsb Hosts Injected 4 days before with Ml@ CD6+ Cells: Enrichment Induced by Normal CD8+ Cells but Not by Irradiated Cells.

Experiment 1.

3d

4d

Sd

Day

14

160

3d

After -

4d

Sd

Injection 80-

2.

140 ^

120

e

100 I

x

^ b

80

-2

60

60-

40f

i 4

40 f 20 O-:

*

3d

4d

5d

-

-.-.-.-m.-.-“‘.l.

3d

Figure 3. Accelerated MLR by CD4+ Cells Recovered Given Ml9 Cells 4 Days Before

4d

Sd

BlO.BR Injected

Mice with:

Uninjected 5 x 106AKR/J CD8+ T 5 x l@AKR/J CD8+ T 5 x lO’+AKR/J CD6+ T Uninjected 2 x lO’AKR/J CD8+ T 2 x 10’AKRlJ CD8+ T 2 x 10sAKRN CD8+ T 2 x 106AKR/J CD8+ T

Percent

of CD4+ Cells

Irradiation of lniected

Expressing:

Cells

‘436

‘43’3

-

9.9 38.6

18.4 15.7

-

20.8

17.4

-

14.0

16.8

-

10.6 32.5

16.2 15.7

+

11.2

16.1

-

21.8

15.5

+

11.8

17.7

Thymectomized BlO.BR mice were injected with various doses of AKR/J CD8+ cells. The percentage V88’ and V68’ cells among CD4+ LN T cells was determined 4 days after injection.

from Mlsb Mice

ATx BlO.BR (Ml@) mice were injected with 10’ (BlO.BR x AKR/J)F, (MIsa) CD8+ cells (triangles) or T- spleen (squares) iv. At 4 days or 14 days after transfer, LN cells were removed from the injected mice and treated with antibody and complement to prepare purified host (Thy l.l-) CD4+ cells. T cells from two individual injected mice together with T cells from uninjected control mice (circles) were tested in MLR for reactivity to Ml.9 (AKFUJ) spleen stimulators (A, C; solid lines), alloH-2p (Bl0.P) stimulators (B,D; dashed lines), or syngeneic (BlO.BR) stimulators (B,D; dotted and dashed lines); responder cells were used at doses of 1.5 x lo5 per well and stimulators (T spleen) at 5 x 10s cells per well. [3H]thymidine was added 18 hr prior to harvest. The responses shown are the mean levels of 13H)thymidine incorporated for triplicate cultures harvested on the days indicated.

cells led to near maximal elevation of VpS+ cells (Figure 2) and even doses as low as 1 x lo5 CD8+ cells caused a P-fold increase in Vf36+ cells (data not shown). T- spleen cells were as potent as CD8+ cells and in some experiments were more potent. Three other features of the early elevation of Vf36+ cells need to be mentioned. First, testing the function of the host CD4+ cells taken on day 4 after injection showed that these cells were clearly MI+reactive in MLR (Figure 3, top). However, this response peaked very early in culture (on or before day 3 of MLR); responses at later stages (days 4, 5) were very low. These abnormal kinetics are characteristic of activated T cells. In contrast to T cells tested on day 4 after injection, the residual anti-MI9 MLR mediated by T cells taken at day 14 did not show accelerated kinetics (Figure 3, bottom). Second, the elevation of Vf%+ cells at day 4 after injection was not observed in recipients of irradiated Ml9 cells, even when these cells were injected in very large numbers (Table 5). Third, the extent of V86+ cell elevation on day 4 was reciprocally

related to the subsequent deletion of these cells at later stages. Thus, Mlsa cell populations eliciting the highest level of Vf36+ cells on day 4 produced the strongest deletion of these cells at 2-3 weeks after transfer (Figure 2). In control, uninjected ATx mice, no such alterations in Vf36 expression were seen. With regard to the question of chimerism, it should be noted that most of the above experiments involved transfer of AKRlJ (Thy-1.1) cells into BlO.BR (Thy-1.2) mice. The Thy-l difference between these strains made it possible to eliminate surviving donor T cells in assessing tolerance and Vf36 expression of the host T cells. With injection of CD8+ or CD4+ cells, small numbers of donor T cells were clearly evident at day 4 after transfer but were scarce at day 7. By day 14 donor T cells were undetectable. Discussion Although the notion that clonal deletion is a major mechanism of tolerance induction is now well established, clonal deletion has only been demonstrated at the level of immature T cells in the thymus; tolerance of mature T cells is considered to be due largely either to suppression or to clonal anergy resulting from defective presentation of antigen. Our data challenge this view by demonstrating that, in certain situations, tolerance of mature T cells can involve clonal deletion. In speculating on the cause of the clonal deletion observed in the present study, it should be stressed that the elimination of Vf36+ cells and functional tolerance seen in Mlsb mice given Ml9 lymphoid cells required the injection of viable cells. Irradiated cells, even in large numbers,

Cdl 1254

were ineffective. Since the injected lymphoid cells included normal B cells, i.e., cells with excellent APC function for MIS-reactive T cells, it is therefore unlikely that tolerance/clonal deletion was related to defective presentation of antigen. In fact, the unexpected finding that the deletion of V86+ cells was preceded by a marked early expansion of these cells indicates that tolerance/deletion was the consequence of a powerful immune response. At face value, the finding that tolerance/clonal deletion can be a consequence of immunity seems paradoxical. Nevertheless, it has long been argued that exposure of lymphocytes to “too much antigen” can eventually lead to hyporesponsiveness. In the 1960s there was considerable interest in the idea that prolonged exposure of lymphocytes to antigen can lead to “exhaustion” or “terminal differentiation” (Simonsen, 1962; Sterzl and Silverstein, 1967; Byers and Sercarz, 1966). Although these early studies dealt primarily with antibody-forming cells, exhaustion was advanced to explain the finding that by certain parameters, e.g., production of graft versus host disease, priming rodents with strong (MHC) antigens can result in diminished responsiveness rather than memory (Simonsen, 1962). Although much of this early evidence on tolerance of mature lymphocytes might now be attributed to suppression, the data in this paper using anti-V8 antibodies to show selective deletion of CD4+ T cells would seem to provide direct support for the concept of exhaustive differentiation. The key question is why strong stimulation of T cells in vivo can lead to rapid elimination of these cells. What happens to the stimulated cells? Experiments of Sprent and Miller (Sprent et al., 1976; Sprent and Miller, 1976a; Sprent and Miller, 1976b) are relevant to this question. These workers examined the fate of a population of H-2-reactive T cells recovered from thoracic duct lymph of irradiated F, mice given parental-strain T cells 4 days before. Although the lymph-borne T cells exhibited powerful antigen-specific effector function in short-term assays, these cells disappeared rapidly after transfer to syngeneic hosts. Some of the T cells homed to the gut and were apparently excreted from the body. Other cells homed in large numbers to the spleen. Based on studies with radiolabeled cells and secondary adoptive transfer, it was concluded that the vast majority of the T blast cells that homed to the spleen survived for only a few days; these cells died in situ and were engulfed by phagocytes. Only a very small proportion of the blasts survived to become long-lived recirculating memory cells. These findings provide a precedent for the disappearance of Vj36+ cells in the present study. One can envision that after proliferating extensively in response to the injected Ml@ lymphoid cells, the host Vj36+ cells rapidly disappeared as a consequence of cell death in situ and migration to the gut. The finding that exposure of mature T cells to MIS antigens in vivo can result in the eventual disappearance of many of the responding cells raises three issues. First, the data would seem to be at variance with the concept of memory, i.e., that contact with antigen in vivo generally leads to hyperresponsiveness rather than tolerance. On this point it should be emphasized that memory is still

poorly understood at a cellular level. Indeed, the recent report that memory cells are comparatively short-lived in the absence of antigen (Grey and Leanderson, 1990) raises the possibility that many of the examples of memory could be attributed to persistence of antigen with constant production of effector cells. In the system described in this paper, the host Vf36+ cells were probably exposed to antigen for only a few days; this follows from the observation that, at least for TAPC, the injected donor cells were undetectable after day 7 after injection. The finding that Mlsareactive Vf36’ T cells disappeared rapidly after stimulation is thus consistent with the notion that memory cells are short-lived. Without additional information, however, the dogma that memory can reflect the activity of cells with a prolonged life span (Gowens and McGregor, 1965) cannot be excluded. Anti-V8 antibodies and clonal populations of T cells from TCR transgenic mice provide important tools for reevaluating the concept of memory. Second, the finding that mature T cells can undergo clonal elimination in vivo should not be taken to indicate that immature and mature T cells are deleted by an identical mechanism. For immature T cells, all of the available evidence suggests that tolerance induction in the thymus is associated with rapid death (apoptosis) without proliferation (Smith et al., 1989; Blackman et al., 1990b). For mature T cells, by contrast, clonal elimination of T cells probably hinges on a prior proliferative response. For this reason, the deletion of immature and mature T cells may be largely unrelated, perhaps reflecting differences in signaling pathways operative in mature versus immature T cells. Third, the observation that tolerance of mature T cells can reflect clonal deletion does not diminish the importance of anergy as a major mechanism of tolerance induction. In future experiments it will be important to establish the precise requirements for inducing anergy versus deletion. One possibility is that anergy depends on persistence of antigen. Certain features of the MIS tolerance observed in the present study require comment. As discussed earlier, the failure of other workers (Rammensee et al., 1989) to observe clonal elimination of VpS+ T cells after exposure to Mlsa antigens in vivo might have reflected de novo production of V86+ cells in the host thymus. We avoided this problem by using thymectomited mice as hosts. Since the elimination of Vj36’ cells in the present study was not complete, the question arises whether the residual nondeleted Vf36+ cells were rendered anergic. When purified by fluorescence-activated cell sorting, these residual cells showed only low (<250/o of normal) reactivity to Mlsa antigens in vitro (unpublished data). Although this finding is consistent with anergy, it is also possible that the nondeleted V86+ cells were a subset of cells with only limited Mlsa reactivity. Thus, it is not clear that all VpS+ T cells have overt Mlsa reactivity. Since the capacity to stimulate Mlsa-reactive T cell clones and hybridomas in vitro appears to be limited to B cells, it is surprising that injecting mice with purified Mlsa CD8+ cells induced the same sequence of Vj36+ cell expansion/elimination as B cells. Although we observed pre-

Tolerance 1255

of Mature

T Cells Reflecting

Clonal

Deletion

viously that Mlsa CD8+ cells are strongly tolerogenic in terms of intrathymic tolerance induction in neonatal mice (Webb and Sprent, 1990), the finding that CD8+ cells caused a marked early elevation of Vp6+ cells when transferred to adult mice indicates that Mlsa antigens on CD8+ cells can be strongly immunogenic. Since responses to MIS antigens involve corecognition of H-2 class II (la) molecules (reviewed in Abe and Hodes, 1989), the fact that la expression on murine T cells is very low makes it difficult to envisage that Mlsa antigens on CD8+ cells are directly immunogenic. A number of groups favor the view that MIS antigens can be recognized in processed form; these antigens elute from MIS= cells, e.g., CD8+ cells, and are displayed on APC in conjunction with la molecules (Pullen et al., 1988; Ramsdell et al., 1989; Speiser et al., 1989). Unequivocal evidence for processing of Mlsa antigens is lacking, however, and it is notable that injection of even high doses of irradiated MIS= CD8+ cells failed to stimulate Vp6+ cell expansion in the spleen, even though homing of T cells to the spleen is not affected by irradiation (Anderson et al., 1974). An alternative possibility is that MIS antigens and la molecules can be recognized on separate cells, e.g., on CD8+ cells and APC, respectively (Webb and Sprent, 1990). This three-cell model has yet to be proved, however, and until MIS molecules are characterized biochemically, the issue of how T cells recognize Mlsa antigens is unlikely to be resolved. In summary, the main finding in this paper is that powerful immune responses in vivo can eventually culminate in marked tolerance and clonal elimination of the responding T cells. This finding has important implications for understanding immunoregulation and the relationship of tolerance and memory. In future studies it will be critical to examine how the consequences of contact with antigendeath and/or memory-are regulated at the biochemical and molecular levels. Experimental Procedures Mice Mice were obtained from either the Scripps Clinic and Research Foundation vivarium (La Jolla, CA) or Jackson Laboratories (Bar Harbor, ME).

Monoclonal Antibodies The following monoclonal antibodies (MAbs) were used in this study: anti-8 cell (Jlld, Bruce et al., 1981); anti-Thy-l.2 (Jij, Bruce et al., 1981); antiThy-1, non-allele-specific (T24, Dennert et al., 1980); anti-CD8 (3.188, Sarmiento et al., 1980); anti-CD4 (RL172, Ceredig et al., 1985; or GK1.5, Dialynas et al., 1983); anti-IAk (10.2.18, Oi et al., 1989); antiVP TCR (RR47, Kanagawa et al., 1989): anti-VW.1 + 8.2 (KJ16, Haskins et al. 1984).

Cell Purifications for InJectIon Cell purifications were conducted as described in Webb and Sprent (1990). Lymph node cell suspensions were passed over nylon wool columns and then treated with MAbs plus complement to remove residual B cells (using Jlld + anti-I-Ak MAb) and T cells of the inap propriate phenotype (using anti-CD4 or anti-CD8 MAb). The resulting CD8+ or CD4+ T cells were further purified by positive panning on plates coated with anti-CD8 or anti-CD4 MAb. This combination of negative and positive selection generated >99% purified populations of CD8+ and CD4+ cells. For T-depleted spleen cell suspensions, spleen cells were subjected to two rounds of treatment with a cocktail of anti-T cell MAb (anti-Thy-l, anti-CD4, and anti-CD8) plus complement. B cells were purified from spleen by passage over GlO columns,

followed by two rounds of anti-T cell MAb plus complement treatment. Small resting B cells were then isolated from the 1.08/1.09 interface of Percoll density gradients. Mlxed Lymphocyte Responses (MLR) MLR were set up as previously described (Webb and Sprent, 1990). Responder T cells were enriched for CD4+ cells by treatment of lymph node cells with Jlld, anti-CD8, and anti-IA MAb plus complement. Approximately 1.5 x lo5 CD4+ cells were cultured with 5 x lo5 mitomytin c-treated spleen stimulator cells depleted of T cells by treatment with anti-Thy-1 MAb and complement. Cultures were pulsed with 1 PCi per well of 13H]thymidine approximately 18 hr before harvesting onto fiber filter-mats, and were then counted in a scintillation counter. Flow Cytometry Analysis of TCR Expresslon Cells for analysis were incubated with an appropriate dilution of MAbcontaining ascites fluid; a fluorescein isothiocyanate-conjugated mouse anti-rat immunoglobulin antibody (Pel Freez, Arkansas) was used to detect the binding of these antibodies. For double staining, phycoerythrin-conjugated anti-CD4 (GK1.5, Becton Dickinson, Mountain View, CA) was used. Thymectomy Mice were thymectomized

as described

by Miller (1960).

Acknowledgments This work was supported by National Institutes of Health grants CA41993, CA25803, CA38355 and A121487. We thank D. W. McQuitty for flow cytometry analysis, J. Hutchinson for excellent technical assistance, and H. Brewer for preparation of the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisemenl’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received

July 26, 1990; revised

October

12, 1990

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