Transplant. Rev. (1975), Vol. 26 Published by Munksgaard, Copenhagen, Denmark No pan may be reproduced by any process without written permission from the author(s)

Suppressor T Cells RICHARD W. DUTTON

The purpose of this review is to summarize our studies on the properties of Concanavalin A (Con A)-induced suppressor T cell activity in the in vitro humoral immune response. We will, in addition, make some attempt to relate this T cell suppressive activity both to the suppressor activity exhibited by T cells in experimental models described by other workers, and to other mitogen-induced T cell activities. A mmiber of phenomena were described during the 196O's, which, in retrospect, can be seen to depend on the activity of suppressor T cells (Gershon 1974), but it was Gershon & Kondo (1970, 1971) who first proposed that T cells were in some way involved in the specific suppression of an immune response. Following these studies, suppressor T cells have been clearly demonstrated in a number of experimental models. The essential feature in each case is that one population of cells will suppress some activity or function of a second cell population in a cell transfer or in vitro experiment. The activity of the suppressor population is stopped by removal of T cells. In addition, the suppression is directed towards some specific function of the supprerssed population, and is not a manifestation of a general cytotoxic effect. Thus, Herzenberg and her colleagues (Herzenberg & Herzenberg 1974) have shown that T cells mediate allotype suppression in the mouse and that the suppression is allotype-spedfic. A number of workers have shown that tolerance to a spedfic antigen can be mediated by suppressive T cells (Basten et al. 1974, Weber & Kolsch 1973). Here the suppression is antigen-specific. Rich & Pierce (1974) have shown that the culture supematants of mitogen-stimulated T cells will suppress the response of fresh cells to all antigens tested. Here the suppression would at least appear to be non-antigen-specific, but the culture supematants are not cytotoxic and do not inhibit other activities of the suppressed cells. In contrast, Okumura & Tada (1974) have obtained a cell-free preparation that Department of Biology, University of California, San Diego, La Jolla, California 92037, U.S.A.

40

RICHARD W. DUTTON

mediates suppression of a specific immune response. In the examples so far, the activity suppressed was the B cell response to antigen and the production of immunoglobulin. In other experimental models, however, one T cell population has been shown to suppress the activities of a second T cell population, such as T cell proliferation (Gershon et al. 1974, Folch & Waksman 1974, Rich & Rich 1975) or the development of T cell cytotoxicity to aliogeneic cells (Peavy & Pierce 1974). It would seem likely that, in these various experimental models, we are studying a number of related suppressor T cell systems that may differ from one another in a number of respects. An additional question may serve to clarify the pohit. How is the specificity of suppression achieved? There are several possibilities. Receptors on the suppressor cell may recognize some specific determinant of the target cell and may then attack that cell in some way as to make it inactive. The specific determinant on the target cell could be antigen bound to the cell surface immunoglobulin receptor, or it could be the idiotype of the receptor itself. In the case of allotype suppression, the target would appear to be allotypic determinants on the immunoglobulin, while the cell-free suppressor supernatant of Okumura & Tada (1974) has antigen binding activity. The specific recognition may occur only at the level of T cell receptor and the suppressor T cell once 'steered' to the target by its receptors could release a non-specific suppressor substance in the immediate vicinity of the target. In this case one would expect the B cell to have special receptors that would recognize the inhibitory signal. Alternatively, the mediator itself might retain antigen specificity as described by Okumura & Tada, but would stUl, presumably, require a second active site to mediate its suppressive effect. It is possible that there are separate receptors on the surface of the B cell for helper and suppressor molecules, and that each produces an 'active' response in the B cell. Alternatively, it is possible that an 'inactive' suppressor mediator merely blocks a single site which Is thus prevented from reacting with the helper mediator (Katz & Benacerraf 1975). The finding that both helper (Annerding et al. 1974, Taussig & Munro 1974, Mimro et al. 1974) and suppressor mediators (Tada, quoted in Benacerraf & Katz 1975) can be removed by passage over insolubilized anti-la lends support to the concept of specialized mediators sendings messages that are received at specific receptors, but other models can certainly be conceived. For example, it has been suggested that suppression can result from the build-up of antigenantibody complexes on the B cell surface (Schrader & Nossal 1974), or from an excessive bombardment by stimulatory signals (Coutinho & Moller, in press). In the discussion above it has been tacitly assumed that suppression of the humoral response is mediated at the level of the B cell itself. Others have

SUPPRESSOR T CELLS

41

argued that suppression is always directed against T cells and that the B cell is denied T cell help by suppression of the helper cell. Such a mechanism is certainly possible and suppression of T cell function has been shown to occur (see above). It would seem not unlikely that both mechanisms exist, and we know of no evidence that would exclude either of them as a means of suppression of B cell function. This concludes some rather brief speculations on the nature of T cell suppression and we will turn now to a summary of our studies on Con A-induced suppressive activity. STUDIES ON CON A-INDUCED SUPPRESSION

In these studies, we have used the in vitro culture system in which we can measure the primary and secondary responses of mouse spleen cells to erythrocyte antigens, hapten-erythrocyte conjugates or hapten-protein conjugates (Mishell & Dutton 1967, Kettman & Dutton 1970, Trefts, manuscript in preparation). The response is measured in terms of the number of direct or indirect plaque-forming cells per culture after 2, 3, 4, or 5 days of culture. The response depends on successful initiation events and on the proliferation and differentiation of the responding cells. There is accumulating evidence that these are separately regulated events and external factors may influence the size of the response at each or any of these points. THE INDUCTION OF SUPPRESSION

When Con A is added to freshly prepared mouse spleen cell suspensions at a density of 1 X 10"^ cells/ml, there is a marked inhibition of the immune response. This is true of both primary and secondary responses, IgM and IgG, and for responses to all the antigens tested (heterologous erythrocytes, TNPSRBC or TNP-KLH). The degree of inhibition is markedly dose-dependent. In medium containing 5 % FBS, the optimal inhibition is seen at 2 /j.g Con A/ml. (Lower concentrations are optimal in the absence of serum). The inhibition of the primary IgM response to SRBC is illustrated in Figure 1. It can be seen (in the same figure) that the dose-response curve for the mitogenic effect, measured either by thymidine uptake in the 80-96 hr culture period, or by cell recovery, is similar. Viability measurements showed that there is an increased proportion of dead cells in the maximally stimulated cultures (Dutton 1972). It is of interest that higher concentrations of mitogen, in the range of 4-8 /zg/ml, are less inhibitory than 2 /tg/ml, supporting the concept that the inhibition depends on some mechanism more subtle than the mere blocking of some

42

RICHARD W. DUTTON

1000- '60

M

1,000

500--30

0

I/It

M

I CON t

i the cell recovery expressed as percent of cells initially; • - # , the incorporation of thymidine-^H present during the last 16 hrs of culture (80-96 hrs) expressed as cpm per culture (Dutton 1972).

Structure on the responding cell or on the antigen. The effect of Con A can be competitively blocked by the simultaneous addition of a-methyl D-mannopyranoside (Figure 2). Kinetic studies revealed that the inhibition effect is not manifested immediately, but becomes marked only on day 3 or 4. Similar observation have been made by Rich & Pierce (1973a, 1973b, 1974). In other experimental models using rabbit spleen cell suspensions, however, the effect of Con A appears to be more immediate, arresting the response at the level it has reached at the time that the Con A is added (Redelman et al. personal communication). One can speculate that effective inhibition may develop earlier if more differentiated suppressor cells are already present when the mitogen as added. When Con A is added at various times after initiation of the culture, it can be seen that the ability to induce an inhibitory effect is rapidly lost and at 24 hours Con A is often stimulatory (Figure 3). The kinetics of this change from inhibition to stimulation are dependent on factors such as cell density and a number of other variables which affect the relative numbers or activity of suppressor and stimulatory cells (see below). In extreme cases, stimulation can be seen at zero time, or inhibition at 24 hours. In the absence of serum. Con A is always stimulatory when added at zero time.

43 so

10^00

1,000

40

I

i

10

ZO

a-HCTHTL D-MANNOPYRANOSIDE

(ng/mll

Figure 2. Prevention of the effects of Con A with tf-methyl D-mannopyranoside. Cell suspensions from the spleens of normal mice were incubated in the presence or absence of 2 /^g/ml Con A and varying concentrations of a-methyl D-mannopyranoside. SRBC were added at T = 0, and the anti-SRBC PFC per culture and the cell recovery, expressed as a percent of the number of cells present initially, were assayed on day 4. O - O . PFC response, no Con A; A " A, PFC response + Con A; • - • , cell recovery, no Con A; A - A, cell recovery + Con A (Dutton 1972).

1000

100

-4

0

4

8

12

16

20

24

TIME OF CON A UDlTION RELATIVE TO ANTIGEN (HOURS)

Figure 3. Time of addition of Con A relative to time of antigen addition. In this experiment antigen was added 2 hrs after the beginning of incubation. # control (Dutton 1972).

44

RICHARD W. DUTTON

The argument will be developed that there are two separate populations of T cells; one induced to suppressive activity and another induced to stimulatory or helper activity. Both these activities are mediated by soluble factors. It is interesting to speculate that helper activity titrates out 'pseudo-linearly* with decreasing numbers of helper cells, or concentrations of helper factor, while suppressive activity is much more rapidly lost. It can be seen, however, that the net effect of Con A addition depends very dramatically on the spleen cell density of the culture (Figure 4). In the experiment illustrated, suppression was observed at 8 X lOVml wliile marked stimulation could be seen at 2 X lOVml and below. This is an important practical consideration for all those engaged in studying such effects.

2

4

CELLS/ml»10-*

Figure 4. Spleen cells were cultured at various cell densities in the presence (A) or absence ( • ) of Con A at 2 fig/ml added at zero time. The cultures were cultured in Falcon Microtest II culture plates and the total volume of culture medium was 0.1 ml per well. 10 /A of 0.05 % SRBC were added at zero time and the number of PFC per well were assayed at day 4. The results are the geometric mean of 12 replicates (T. G. A. Coha, unpublished data).

PROPERTIES OF TEIE SUPPRESSOR CELL

It can be shown that Con A-induced suppressive activity can act on normal spleen cells not themselves exposed to Con A (see Figure 5). Rich & Pierce (1974) have been able to prepare a cell-free culture supernatant from Con Aincubated cells which has suppressive activity. This trans-effect, in which a population preincubated with Con A is assayed by its effect on fresh cells, allows a characterization of the properties of the suppressor cells. It has been shown that the activity is abolished by treatment with antimouse brain-associated antigen (anti-MBA) and complement, either at the start or the end of the preincubation period. The Con A-inducible activity

SUPPRESSOR T CELLS

45

tCOOOp

1,000

-

O.I

C.E

0.3

0-4

FflACTION OF PREINCUBATED CELLS

Figure 5. Effect of adding Con A-treated cells to normal mouse spleen cell suspensions. Spleen cell suspensions from the spleens of normal mice were incubated with or without 2 /.ig/ml Con A for 48 hrs. They were then washed twice in 10 ml of sterile BSS and resuspended in complete medium at 1 x lOVml. Graded amounts of the resulting suspensions were added to suspensions of fresh normal mouse spleens keeping the total cell number constant at 1 X lOVml. SRBC were added at T = 0 and the anti-SRBC PFC response was measured at day 4. Cells preincubated with Con A ( O - O ) aiid without (A - A) (Dutton 1972).

cannot be observed when Con A is added to spleen cells from adult thymectomized, irradiated, bone marrow restored mice (Figure 6). The Con A-induced suppressor cell is clearly a T cell. The suppressor population is radiosensitive if irradiated prior to preincubation, but radioinsensitive if irradiated after preincubation. Similar properties have been described by Rich & Pierce (1973b). We have also shown that suppressive activity is developed equally effectively if adherent cells are removed either prior to or after the preincubation period (McCarthy & Dutton, unpublished observations). The suppressor cell activity is absent from the spleens of Nu-Nu mice (Dutton 1973) and disappears progressively from the spleens of adult thymectomized mice (Kappler & Marrack, in press). The kinetics of the disappearance are complex. Approximately half of the activity disappears with a short half-life (2-3 weeks), while the remainder is much more persistent. Con A-inducible suppressive activity develops only slowly after birth when as-

RICHARD W. DUTTON

46

©

0^00

PFC RESPONSE —O

1,000

J too

- 80

CELL RECOVERIES

=e - 40

10 •

1

\ ,,

0

1

1

1/16

1/4

1

CON A

Figure 6. Effect of Con A on the response of four ATxBM mice spleens to SRBC. Spleen cell suspensions from four individual ATxBM mice were incubated with SRBC and various concentrations of Con A added at T = 0. The number of PFC/culture and the cell recoveries were assayed at day 4. The results obtained with four separate mice are illustrated A , O . O, A (Dutton 1972).

sayed in spleen cell suspensions (Table 1). It is present in mesenteric lymph nodes and in thymus. Cells with suppressive activity co-sediment with large cells and cells labeled with H^ thymidine when cells preincubated with Con A for 40 hours are sedimented on a FicoII velocity gradient (Tse & Dutton, manuscript in preparation and Figures 7(a) and (b)). TABLE I Responses of young mice

No addition Con A at Ohrs Con A at 24 hrs

2wks

5 wks

8 wks

35 50 410

60 165 735

555 465 3200

5 X 10* spleen cells from BDFj spleen cells from BDFj mice of various ages were incubated in one milliliter of medium. SRBC were added at t = 0. Con A was added at the times indicated at a final concentration of 2 piz/rrA. The PFC/culture were assayed at day 4. (Dutton 1975.)

SUPPRESSOR T CELLS

5.5

1

'

n

' m .

IS

47

'

5.0

\ \

4,5 4.0 • _

3.5

^30 "E

-2.5

/

1.5 1.0 0.5

-IS

M

J 4 \

2.0

/A /// 10

15

20

25

FRACTION NUMBER

^•

\• 30

34

0 0.5 I D

E.0

3.0

4.0

, NUMBER OF FRACTIONATED CELLS ADDEDWO-

Figure 7(a). Profile of cell distribution and 'H thymidine incorporation. BDF^ spleen cells, cultured with Con A, were labeled with ^H thymidine from 20-40 hrs. A FicollHypague gradient was used to remove dead cells and the subsequent cell suspension was irradiated with 1000 R. 1 ml containing 4 X 10' cells was layered on a continuous 5 %20 % Ficoll gradient. The gradient was 30 ml with 3 ml of 5 % Ficoll layered on top to prevent 'streaming'. The cells were centrlfuged for 30 min at 100 g. 1 ml fractions were then taken from the gradient for assay. In one experiment, the number of cells in each fraction was determined. In a second experiment, 0.1 ml from each fraction was assayed for ^H thymidine incorporation and the rest was pooled into fractions I, U, III and IV as indicated. Various numbers of cells from each pooled fraction were then titraded into a fixed number of normal spleen cells as in Figure 7(b) to assay stimulator and suppressor function. A - A , cpm 'H thymidine (X 10"'; • - • cell number/fraction; % Ficoll. (Tse & Dutton, manuscript in preparation.) Figure 7(b). Effect of fractionated cells on the normal response of spleen cells to SRBC. Various number of fractionated and unfractionated cells were cultured with 10* BDFj^ spleen cells and SRBC in microtiter plates. The hemolytic plaque assay was performed on day 4. Each point represents the arithmetic mean of PFC from 8 replicate wells. (Tse & Dutton, manuscript in preparation.)

CON A-INDUCED STIMULATORY ACnVITY AND THE PROPERTIES OF STIMULATOR CELLS

It has been shown above that Con A-inducible suppression is abrogated by any of a number of procedures. Thus, irradiation, treatment with anti-MBA, brief incubation in culture prior to the addition of Con A, use of spleen cells from ATxBM mice, or just by using cell cultures at lowered cell density, or in the absence of serum, leads to a loss of suppressive activity and the emergence of a Con A-induced stimulatory effect. One example of such an eSect

RICHARD W. DUTTON

48 lODOO

^

100

10

NOKE

1144

1.72

1:36

1.16

19

DILUTION OF GOAT ANTI-BA THYHUS ANTIGEN

Figure 8. Titration of the efEect of goat anti-BA thymus antigen on the response of spleen cell suspensions to SRBC in the presence (A - A) and ahsence (O - O) of Con A (Dutton 1972).

is illustrated in Figure 8. This shows that progressively higher concentrations of anti-MBA are increasingly efEective at reducing the immune response (by removing helper T cells). At the same time, however, the cell suspensions cease to be inhibited by Con A and instead are increasingly stimulated. It is clear that the Con A-induced stimulatory activity is very resistant to removal with anti-T cell antiserum and the question can be raised as to whether it is a property of T cells at all. The stimulatory activity is not present in the spleen cells of Nu-Nu mice and the experiment illustrated in Table II shows that irradiated normal spleen cell suspensions preincubated with Con A will restore the immune response to untreated Nu-Nu spleens. Pretreatment of the Nu-Nu spleen cells with Con A, rather than the normal spleen cells in the same experimental model, was without effect. The stimulator cell would thus appear to be a T cell. This 'trans' experimental model, in which irradiated cells preincubated with Con A stimulate the response of Nu-Nu spleen cells to antigen, once again allows a characterization of the Con A-inducible cell. The stimulator cell is much more radio-resistant than the suppressor cell, but its activity is nevertheless reduced to some extent (Figure 9). Stimulatory activity persists for long periods of time in adult thymectimized mice (Kappler & Marrack, in press) and is even present in the spleens of ATxBM mice (see above). It is

SUPPRESSOR T CELLS

49

TABLE n Preincubation of Con A with spleen cells from nude mice or with irradiated spleen cells from BDFj mice Anti-SRBC PFC/ciUture (day 4) Exp. (A) Con A preincubated with nudes (B) Con A preincubated with irrad.; BDFj (C) Con A present entire time nude -I- BDFj irrad. (D) Nude alone (E) BDFj irrad. alone

110

161

162

167

15 175

20 95

0 80

25 370

770 15 0

685 95 0

295 20 0

255 30 0

In (A) 5 X 10* spleen cells from nude mice were incubated with 2 /j.g Con A in 1 ml culture medium for 24 hrs. The cells were then washed twice in 50 ml sterile BSS and then incubated for a further 3 days with 5 X 10* irradiated spleen cells from BDFj mice. In (B) the same procedure was followed except that it was the irradiated BDF^ spleen cells that were exposed to Con A during the first 24 hrs. In (C) 2 /ig/ml Con A was incubated with 5 X 10* nude spleen and 5 X 10* irrad.: BDFj spleen for the entire 4 days. (D) and (E) are the controls, 1 X 10' nude spleen or 1 X 10' irradiated spleen incubated alone. (Dutton 1973.)

10,000 r

ipoo

P

100

z

NONE

3R10«

10^

3x10^

10*^

3x10*

NUMBER OF Nu/t CELLS ADDED

Figure 9. Effect of irradiation dose on the restoration of the response of nude spleen. The details of this experiments are the same as those described for Figure 10, except that in this experiment the added spleen cells were from heterozygous nude mice instead of BDFi. These added cells received zero R (O). 200 R (A), 400 R (V), or 800 R O All cultures contained 8 X 10* nude spleen cells + 2 /ig/ml Con A (Exp. 184) (Dutton 1973). Transplant. Rev. (1975), Vol. 26

50

RICHARD W. DUTTON TABLE n i Development of Con A-induced stimulatory activity in the spleens of young BDFj (C57BU6 X DBA/2) mice

Con A Irradiated spleen BDF^

— -

+ -

Response (PFC/culture)

0 0 0 1 0

0 0 0 0 0

+

+

Newborn l w k 0 0 0 0 0

0 0 0 0 1

+

+1

2wk

5wk

8wk

0 0 0 0 0

10 14 24 4 9

68 28 82 96 74

All cultures contained 2 X 10* spleen cells from Nu-Nu mice. SRBC (3 X 10*/ culture) were added at t = 0 and the response was assayed at day 4. Con A, where present, was at 2 /ig/ml. The irradiated cells received 1000 R. (Dutton 1975.)

difficult to quantify the absolute amount of stimxilatory activity since all that can be measured is the balance of stimulation and suppression. Both stimulatory and suppressive activity may decline after thymectomy but stimulation would appear to persist if the decline of suppressive activity was the more rapid. Con A-inducible stimulatory cells are present in the mesenteric lymphnode population, but appear to be absent or minimal in thymus. Stimulatory activity appears only slowly after birth (Table III). Stimulatory activity is associated with small lymphocytes which remain at the top of a Ficoll velocity sedimentation gradient when cells preincubated with Con A for 40 hours are sedimented (Figures 7(a) and (b). Large cells, suppressor cells, and proliferating cells sediment more rapidly (Tse & Dutton, manuscript in preparation). SEPARATE CELLS ARE RESPONSIBLE FOR STIMULATORY AND SUPPRESSIVE ACTIVITY

It has been suggested that the stimulatory activity is merely the manifestation of small amoimts of suppressive activity, i.e. suppression is merely an excessive amount of stimulation (Coutinho & Moller, in press). This hypothesis has the merit of simplicity and is by no means excluded by much of the foregoing experiments which at first sight would appear to define difEerent properties for the two cell activities. The properties of the two cell activities are summarized in Table IV. It could be argued that all of tbe procedures that remove suppressor activity and reveal stimulatory activity merely reduce the numbers of a single cell type from the suppressive to stimulatory level.

SUPPRESSOR T CELLS

51

TABLE IV Summary of the properties of inhibitory and stimulatory activities Inhibitory activity Present in: 1) Normal spleen 2) Antiserum-treated^ 3) Normal lymph node 4) ATxBM 5) Adult thymectimized 6) Nude spleen 7) Normal spleen preincubated for 24 hrs Sensitive to irradiation PHA stimulatable Sedimentation in FicoU gradient*

yes no yes no absent?^ no no yes yes fast

Stimulatory activity

yes yes yes yes yes no yes no^ no slow

* In the original studies (1) the antiserum used was goat anti-mouse brain. This completely eliminated carrier specific helper activity and Con A induced inhibitory activity but had no apparent effect on the Con A-stimulatory activity. More recent studies with a rabbit anti-mouse brain serum (unpublished) showed that treatment with this antiserum does cause some reduction in Con A-induced stimulatory activity. ^ See text and Kappler & Marrack (in press). 3 Irradiation of normal spleen cell cultures shifts the balance from inhibition to stimulation. The inhibitory activity is very radiosensitive while the stimulatory activity appears to be only slightly affected. It has not been possible to determine the true radiosensitivity of the separate activities (see text). * See Fig. 7a and b for further details. (Modified from Dutton 1975.)

A number of arguments have been presented against such a concept (Dutton 1973,1975, Scavulli & Dutton 1975) and can be summarized briefly here. First, in the experiment where the response of Nu-Nu spleen cells was restored by the addition of varying amounts of irradiated or unirradiated cells in the presence of Con A, there was no concentration of unirradiated cells which would restore the response to as high a level as seen when irradiated cells were used (Figure 10, see also Figure 9). Secondly, phytohemagglutinin (PHA) also induced suppressive activity but did not stimulate stimulatory activity (Dutton 1973). This argument is only valid if it can be assumed that it was the same suppressor cell activity that was stimulated by both mitogens. Thirdly, low concentrations of suppressor supematants did not stimulate the response that would be expected if there were only a single mediator (Rich & Pierce 1974). Finally, the progressively-increasing inhibition obtained when

RICHARD W. DUTTON

52

increasing numbers of suppressor cells are added can be reversed when stimulatory cells are added (Figure 11 and Scavulli & Dutton 1975). If stimulatory cells and suppressor cells were one and the same, the addition of more cells would only have resulted in further suppression.

800 R

1.000

K

100

10 (O)

NUDE ALONE

-6

, 10^ 10* 10* 10* NUMBER OF BDFI CELLS ADDED

Figure 10. Restoration of the response of nude spleen with Con A-stimulated cells. All cultures contained 8 X 10* spleen cells from nude mice, SRBC, and 2 ^g/ml Con A. To these were added graded numbers of irradiated (A) or unirradiated (O) spleen cells from BDFj mice. The irradiation dose was 800 R. The numbers of anti-SRBC PFC per culture were assayed on day 4. Control cultures of 8 X 10* nude spleen cells in the absence of Con A developed 65 PFC/culture by day {Exp. 171) (Dutton 1973).

PHYSIOLOGICAL SIGNIFICANCE ON THE CON A-INDUCIBLE SUPPRESSOR AND STIMULATORY ACTIVITY

It is here that we rely mainly on faith. We incline to the working hypothesis that there are present, in the intact animal, populations of both suppressor and helper cells. These may be newly-formed Tl cells or more differentiated T2 cells that have arisen subsequent to antigen exposure. Both types of cell are antigen-specific by virtue of cell surface antigen receptors. They can be activated by exposure to specific antigen, in which case only the subset of cells specific for that antigen are activated, or they can be activated by mitogens, in which case cells of all antigen-specificity are activated. Thus, we regard mitogen activation of cells as the unphysiological activation of the whole

SUPPRESSOR T CELLS

53

-4 muicH STimiLJiTeii cms .JMILLlim

UMO -

INHIBITOR CELLS x IO~

Figure 11. 3 X 10* Nude spleen cells were incubated with varying numbers of stimulator and suppressor cells. The numbers of stimulator cells are indicated on the individual curves and the number of inhibitor cells on the X axis. The number of anti-SRBC PFC per culture were determined on day 4 (Scavulli & Dutton 1975).

population of cells but that these cells, once activated, carry out their nonnai physiological activities. They carry out their effector functions by the release of non-specific mediators that can act on all B (or T) cells. We do not exclude the possibility that they may retain antigen binding activity and thus be concentrated on cells binding that antigen. The Tl -»- T2 maturation involves extensive proliferation and induction of effector activity from Tl cells and is radiosensitive. The activation of T2 cells involves less (or no?) proliferation and is relatively radio-insensitive. Induced effector cells are radio-resistant. On this model we would have to presimie that there are sufficient T2 stimulator cells to provide for the observed 'T2 type' properties of stimulatory activity. In the unprimed mouse, the suppressor activity appears to reside mainly in the Tl pool. We have obtained some preliminary data that suggest that T2 type suppressor cells can be detected in primed mice (Robinson & Dutton, unpublished observations). Our experiments so far are compatible with this model and we feel justified in continuing to use mitogen activation as a probe to investigate and to characterize the cells involved in these activities in a variety of experimental models.

54

RICHARD W. DUITON

ACKNOWLEDGMENTS

This work was supported by grants from the National Institutes of Health (USPHS AI 08795), and the American Cancer Society (ACS IM-IH and PRA-73). I am indebted to my colleagues for their helpful discussions and criticisms and to Sara Albanil for excellent technical assistance in the experiments described. REFERENCES Amerding, D., Sachs, D. H. & Katz, D. H. (1974) Activation of T and B lymphocytes in vitro. Ul. Presence of Ia determinants on allogenic effect factor (AEF). /. exp. Med. 140, 1717. Basten, A., Miller, J. F. A. P., Sprent, J. & Cheers, C. (1974) Cell to cell interaction in the immune response. X. T-cell dependent suppression in tolerant mice. J. exp. Med. 140, 199. Benacerraf,B. & Katz,D.H. (in press) The nature and function of histocompatibilitylinked immune response genes. In: Advance In Cancer Research, Academic Press Inc., N.Y. Coutinho, A. & Moller, G. (in press) Thymus-independent B cell induction and paralysis. Adv. Immunol. Dutton, R. W. (1972) Inhibitory and stimulatory effects of Concanavalin A on the response of mouse spleen cell suspensions to antigen. I. Characterization of the inhibitory cell activity. /. exp. Med. 136, 1445. Dutton, R. W. (1973) Inhibitory and stimulatory effects of Concanavalin A on the response of mouse spleen cell suspensions to antigen, n . Evidence for separate stimulatory and inhibitory cells. /. exp. Med. 138, 1496. Dutton, R.W. (1975) Mitogens and T cell heterogeneity. Ann. N.Y. Acad. Sci. 249, 43. Folch, H. & Waksman, B. H. (1974) The splenic suppressor cell. II. Suppression of the mixed lymphocyte reaction by thymus dependent adherent cells. /. Immunol. 113, 140. Gershon, R. K. (1974) T cell control of antibody production. In: Contemporary Topics in Immunology 3, eds. Cooper, M. D. & Warner, N. L., pp. 1. Plenum Press, N.Y. Gershon, R. K. & Kondo, K. (1970) CeU interactions in the induction of tolerance. The role of thymic lymphocytes. Immunology 18, 723. Gershon, R. K. & Kondo, K. (1971) Infectious immunological tolerance. Immunology 21. 903. Gershon, R. K., Lance, E. M. & Kondo, K. (1974) Immunoregulatory role of spleen localizing thymocytes. /. Immunol. 112, 546. Herzenberg, L. & Herzenberg, L. (1974) Short term and chronic allotype suppression in mice. In: Contemporary Topics in Immunology 3, eds. Cooper, M. D. & Warner, N. L., pp. 41., Plenum Press, N.Y. Katz, D. H. & Benacerraf, B. (1972) The regulation influence of activated T cells on B cell response to antigen. Adv. Immunol. 15, 2. Katz, D. H. & Benacerraf, B. (1975) Hypothesis: The function and interrelationships of

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