Regulation of cytotoxic responses to alloantigens by Ia+ cells

CELLULAR

IMMUNOLOGY

Regulation

62,82-92 (1981)

of Cytotoxic

Responses

to Alloantigens

by la+ Cells’

H.-S. TEH, S.-J. TEH, AND M. Yu Department

of

Microbiology,

University

Received November

of British

Columbia,

Vancouver. B.C., V&r 1 Wj, Canada

25. 1980; accepted February

22, 1981

Pretreatment of responder spleen cells with anti-Ia plus complement led to an enhancement of cytotoxic responsesto alloantigens as well as to TNP-modified self antigens. This observation confirms previous reports that cytotoxic T lymphocytes (CTL) and their precursors (CLP) are Ia-. Furthermore, it suggests that the CTL responses to alloantigens or TNP-modified self-antigens are regulated by an Ia+ suppressor cell. Absorption studies and studies with antiIa sera specific for either the entire I region or the I-E/C subregions suggest that the regulatory cell certainly expressesI-E/C-coded determinants although the possibility that it also expresses I-A/B/J-coded determinats cannot be ruled out. Cell-mixing studies suggest that the regulatory cell is Thy-l- and requires cell division before it can suppress. A clonal assay for CLP was used to show that the enhancement of the CTL response to alloantigens cannot be accounted for on the basis of an increase in the number of CLP in the anti-Ia + C-treated group.

INTRODUCTION Antisera specific for Ia antigens have proved useful in the identification of functional subpopulations of B cells ( 1), macrophages (2) T suppressor cells (3), and T helper cells (4). The existence of Ia antigens on cytotoxic T lymphocytes (CTL)2 is more controversial. In vivo studies indicated that the successful interaction between helper and cytotoxic T cells specific for virus or male antigen- modified syngeneic cells depended upon the ability of helper cells to recognize putative I-A products on the CTL precursors (CLP) (5, 6). However, studies with conventional anti-Ia sera have shown that effector CTL specific for alloantigens (7) or virusmodified self-antigen (8) are Ia-. Pretreatment of the responder cells with anti-Ia serum and complement (C) also has no effect on CTL responses to alloantigens (9). In contrast, Plate (10) reported that pretreatment of lymph node lymphocytes with anti-Ia plus C abolished the capacity of such cells to develop MLR and also the capacity to generate CTL in vitro. Fujimoto et al. (11) also reported that CTL specific for some, but not all, syngeneic tumors bear Ia determinants. In view of these conflicting reports we decided to reexamine the Ia phenotype of CLP to alloantigens or trinitrophenyl (TNP)-modified self-antigens. Unexpectedly, we found that the cytotoxic responses to alloantigens or TNP-modified self-antigens were significantly enhanced when the responding population was treated with con’ This work was supported by the National Cancer Institute and the Medical Research Council of Canada. ’ Abbreviations used: CTL, cytotoxic T lymphocytes; CLP, precursors of CTL; C, complement; Con A, concanavalin A; Ia, I region associated; IL2, interleukin 2; TNP, trinitrophenyl. 82 0008-8749/81/l 10082-11$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form rcscrvcd.

REGULATION

OF CTL RESPONSES BY Iaf CELLS

83

ventional anti-Ia sera + C. This suggests that the CLP are not only Ia- as previously reported by Lonai (9) but also suggests that cytotoxic responsesto alloantigens or TNP-modified self-antigens are regulated by an Iaf cell. The regulation of cytotoxic responseto minor histocompatibility antigens by Ia+ cells has been reported recently by Hayes et al. ( 12). The significance of our findings with regard to current models of CTL activation and regulation is discussed. MATERIALS

AND METHODS

Mice. C57BL/lO, BlO.BR, BlO.D2, BlO.A, BlO.HTT, BlO.A(3R), and A.TH mice were bred in the animal facility in this department. Both male and female mice were used but within a given experiment, only male or only female mice were used. Anti-la sera. A.TH anti-A.TL (anti-Iak) serum, with specificities for I-(A/B/J/ E/C)k, Tla” was provided by the National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. BIO.S (7R) anti-BlO.HTT (anti-I-EkCk) serum was provided by Dr. T. Delovitch of the Best Institute in Toronto, Canada. Anti-Thy1 serum was a gift from Dr. M. Letarte of the Division of Immunology, Hospital for Sick Children, Toronto, Canada. It was prepared by immunizing rabbits with purified mouse brain Thy-l glycoprotein (13). For treatment of cells with these antisera, up to 5 X 10’ spleen cells were incubated with the appropriate dilution of antiserum (usually l/10 for anti-Ia and l/50 for anti-Thy-l; dilution was made with RPM1 1640 buffered with 10 mM Hepes, pH 7.2) for 40 min at room temperature. The cells were washed once and then incubated with 1 ml of low toxic rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada), diluted six times with RPM1 1640 + 10 mM Hepes, for 40 min at 37°C. The cells were washed three times with culture medium before use. Cell cultures. The culture medium used was RPM1 1640 supplemented with 5 X 1O-’ M 2-mercaptoethanol, 10% fetal bovine serum (GIBCO), and 10 mM Hepes buffer, pH 7.2. For determination of stimulation indices in MLR, 5 X lo5 responder spleen cells were cultured with 5 X 10’ stimulator spleen cells (irradiated with 2000 rad from a “Co source at 32 rad set) in flat-bottom microtiter trays (Linbro ISFB-96-TC) in a total volume of 0.20 ml. The cultures were harvested at 72 hr and 0.5 &i of [3H]thymidine in 50 ~1 of culture medium was added to each culture 6 hr before harvest; the final concentration of thymidine in each culture was 10 PM. Each culture was collected onto a glass fiber disk using a 12-sample harvester. The cultures were washed six times with about 0.3 ml of distilled water each time. The filters were then rinsed with methanol and dried in a 100°C oven for 20 min before being counted in 3 ml of scintillation fluid (4 g PPO/liter toluene) in a Nuclear Chicago Unilux II scintillation counter. Stimulation indices were expressed as the ratio of experimental counts to control counts. For generation of CTL, cultures were set up in Linbro trays (FB-16-24TC) as previously described (14). Unless indicated otherwise, each culture contained 4 X lo6 responder and 2 X lo6 irradiated (2000 rad) stimulator spleen cells in 2.5 ml of medium. The cultures were set up in triplicates and incubated for 4 days at 37’C in a humidified incubator containing 5% CO, in air. Modification of stimulator cells with trinitobenzene sulfonic acid was performed as described by Shearer ( 15).

84

TEH, TEH, AND

YU

CLP frequency estimations. This was set up as previously described ( 16). Briefly, limiting number of responder lymphocytes were cultured with 1 X lo4 irradiated (2000 rad) stimulator spleen cells and 10 units ml-’ costimulator in a total volume of 0.20 ml. Costimulator is a lymphokine obtained by stimulation of spleen cells with concanavalin A (Con A) and is prepared as previously described ( 17). A unit of costimulator activity is defined as the reciprocal of the dilution of costimulator needed to yield 37% of the maximal DNA synthetic responseof thymocytes cultured at 5 X lo5 cells ml-’ with 2 pg ml-’ Con A (18). The cultures were assayed for CTL responses after a 5-day culture period. Target cells. Con A blasts were obtained by culturing 1 X 10’ normal spleen cells with 2 pg ml-’ Con A in a total volume of 10 ml of medium in Falcon tissue culture flasks (No. 30 12) kept in an upright position. The blasts were used as target cells after 2 or 3 days in culture. P8 15, a DBA/2 (H-2d) mastocytoma, was kept as a cultured cell line. The target cells were labeled with Naz5’Cr04 as previously described (14) and the radioactivity was measured in an LKB Rack Gamma II Counter. Spontaneous release for Con A and P815 targets ranged from 15 to 30% and from 8 to 15%, respectively of the maximum release values during a 4-hr assay. Cytotoxity assays. The cytotoxic activities of the cultured cells were determined with the method previously described (14). Briefly the cultures were pooled, washed once, and suspended in an appropriate volume of culture medium. Five serial twofold dilutions of each group were then made in round-bottom microtiter trays (Linbro IS-MRC-965(MR-5); 2 X lo4 “Cr-labeled target cells were then added to each well and the trays were centrifuged at 170g for 5 min at room temperature before being incubated for a further 4 hr at 37°C. The fraction of target cells lysed, f, in each well is then fitted to the equation f= 1 - epNorr(19), where N = total number of sensitized cells, (Yis a constant proportional to the frequency of CTL, and t is the assay time in hours. Under appropriate conditions,fis the same as the fractional specific lysis, p, so that Nat = -1n (1 - p). P is defined as experimental counts - spontaneous counts maximum release - spontaneous counts ’ Maximum release was determined by freezing and thawing the targets three times. The advantage of reexpressing specific lysis values in this way is that they are now directly proportional to the number of CTL present rather than the number of target cells destroyed. This then allowed a direct comparison of the relative cytotoxic activities for the cultures assayed. Cytotoxic assays for limiting dilution cultures were performed as previously described (16). After a 5-day culture period, 0.10 ml of cultutre supernatant was removed from each culture and 2 X 10’ “Cr-labeled target cells in 0.10 ml of culture medium was added to each culture, suspending the cell pellet in the process. The cultures were then centrifuged at 170g at room temperature for 5 min and incubated for a further 41/2 hr at 37°C; 0.10 ml of supernatant was then sampled and counted for 2 min in a Picker PACE-l gamma counter. Spontaneous release was determined by taking the mean of the “Cr counts from 12 cultures that received stimulator cells and costimulator but no responder cells. A culture was scored as positive if its counts exceeded the 95% confidence limits of the spontaneous counts; a positive culture usually corresponds to greater than 5% specific lysis.

REGULATION

85

OF CTL RESPONSES BY la+ CELLS

The frequencies of CLP in the responding population to the stimulating antigen were calculated by Poisson statistics according to the method of Porter and Berry (20). This method of calculation weighs each experimental point and calculates the 95% confidence limits of each CLP frequency estimation. Furthermore, it provides an indication of whether the experimental values are consistent with Poisson statistics by calculating the x2 value of each frequency estimation. \

RESULTS

The la phenotype of stimulator and responder cells in MLR and cytotoxic responses. BlO.BR spleen cells were treated with A.TH anti-A.TL serum and C.

This treatment led to a specific killing of between 40 and 55% of BlO.BR spleen cells over that of the C control. After treatment the cell density was readjusted such that each culture received the same initial number of viable lymphocytes. The ability of the treated cells to function as a stimulator and as a responder in MLR was first examined. Four such experiments were performed and the results of one typical experiment are shown in Table 1. In agreement with previous studies (9) it is noted that the BlO.BR spleen cells which had been treated with anti-Ia + C had a greatly reduced ability to stimulate in an MLR (lines 3 and 4, Table 1). The ability of the anti-Ia-treated population to stimulate weakly (S.I. = 2.12) could be due to stimulation by H-2K/D coded antigens since it is known that H-2K/D antigens can stimulate weakly in an MLR (21). Alternatively, this weak stimulation may be due to incomplete removal of Ia+ cells. On the other hand, the treated cells were fully capable of responding in an MLR (lines 7 and 8, Table 1). Similar results were obtained for different concentrations (1 in 50 to 1 in 10) of anti-Ia serum (data not shown). In two experiments, although the ability of the anti-Iatreated cells to stimulate in an MLR was completely abrogated, they retained the ability to respond proliferatively in an MLR. These data are in agreement with those reported by Lonai (9) and suggest that the responder cells in MLR are Ia-. TABLE 1 The Ia Phenotype of Stimulator and Responder Cells in MLR” Responder 5x 5x 5x 5x

Treatmentb

IO5 BlO lo5 BIO IO5 BIO IO5 BIO

None None None None

5 X 10’ BIO.BR 5 X IO5 BIO.BR 5 X lo5 BlO.BR 5 X lo5 BlO.BR

None None C C + cuIa’

Stimulator (2000R) 5 X 10’ BIO 5 X IO5 BlO.BR 5 X IO’ BlO.BR 5X 5X 5X 5X

10’ BIO.BR IO’ BIO lo5 BlO IO5 BlO

Treatment

Stimulation Index’

None None C C + aIak

1.00 f 0.10 7.45 + 0.32 7.16 f 0.39 2.12 zk 0.07

None None None None

1.00 3.72 4.05 5.66

f + k f

0.05 0.10 0.17 0.15

’ Cultures were set up and assayed as described under Materials and Methods. b Spleen cells were treated with A.TH anti-A.TL serum (diluted 1 in 10) + C as described under Materials and Methods. ’ Mean -+ SE for five cultures. Control counts were 2306 + 231 and 2544 f 115 for BIO and BlO.BR, respectively.

86

TEH, TEH, AND

YU

The anti-Ia + C-treated cells were also tested for their ability to act as stimulators or responders in cytotoxic responsesto alloantigens. (Table 2). The Ia- spleen cells showed about a twofold reduction in their ability to act as stimulators (lines 1 to 3, Table 2). This reduction was less than that observed in an MLR. Thus, Ia+ stimulators appear not to be essential for the induction of a cytotoxic response but have an enhancing effect on an ongoing cytotoxic response as previously reported (22). On the other hand, the anti-Ia-treated cells responded 9.4 times better to H.2b alloantigens than the C-treated cells. Since the anti-Ia-treated group received only 1.7 times more Ia- cells than the complement treated group in this experiment, one cannot account for this elevated response even if the CTL responsewas directly proportional to the number of Ia- CLP present in each culture. We conclude from these data that the CLP is not only Ia- as previously reported (9) but also that cytotoxic responses to alloantigens are controlled by an Ia+ suppressor cell. Properties of the regulatory cell. The augmented CTL responsesto alloantigens in the anti-Ia-treated population were observed over a range of cell densitites tested (Fig. 1). In the anti-Ia-treated group the CTL response did not start to decrease until the cell number reaches 8 X lo6 cells/culture whereas in the C control group, the cytotoxic response started to decrease by 6 X IO6 cells/culture. The simplest explanation for these data is that the anti-Ia treated group has fewer Ia+ suppressor cells than the C control and consequently was less sensitive to suppression as a result of an increase in cell number. Cell mixing studies suggest that the regulatory cell is radiation sensitive and Thy-l- (Fig. 2). Thus, the addition of irradiated normal spleen cells to the anti-Ia-treated group did not result in a decrease of the overall cytotoxic response whereas the addition of C-treated or anti-Thy-l + Ctreated spleen cells result in a suppression of the overall CTL response. These studies suggest that the regulatory cell is probably Thy-l- and requires cell proliferation before it can suppress. However, since the suppressive effect was observed only after the addition of a large number of cells this conclusion is at best tentative. A more sensitive culture system capable of detecting small numbers of suppressor cells is necessary to confirm these results. Mapping of the la determinants on the regulatory cell. The enhancing activity TABLE The Ia Phenotype of Stimulator

2

and Responder Cells in Cytotoxic

Responses to Alloantigens” Lytic activity

Responder 4 x lo6 BIO 4 X IO6 BIO 4 x IO6 BlO 4 x IO6 BlO.BR 4 x lo6 BIO.BR 4 x lo6 BIO.BR

Treatment

Stimulator (2000R)

None None None

2 x lo6 BIO.BR 2 x IO6 BlO.BR 2 X IO6 BIO.BR

None C C + cula’

2 x IO6 B10 2 x IO6 BIO 2 x IO6 BIO

Treatment None C C + cuIa’ None None None

BlO target ‘
(Nolt)/culture” BlO.BR target 38.8’ 44.0 20.0
0 Cultures were set up and assayed as described under Materials and Methods. b Calculated according to the method of Miller and Dunkely (19). ’ The specific lysis values from which the Ncvf value of 38.8 was derived were 74, 62, 37, 23, and 12% for effecter/target ratios of 7.2, 3.6, 1.8, 0.9, and 0.45: 1, respectively.

REGULATION

120

OF CTL RESPONSES

BY Ia+ CELLS

87

I

0’

2x10~ No.

of

6~10~

4x106 Responder

cells

per

6~10~ culture

FIG. 1. Effect of varying cell density on cytotoxic responses to alloantigens. BlO.BR (H-2’) spleen cells were treated either with C or A.TH anti-A.TL (anti-Ia’) serum + C. The indicated number of viable cells in each group were then cultured with 4 X lo6 irradiated C57BL/lO spleen cells for 4 days and assayed for CTL activity against 2 X 10’ “0-labeled C57BL/lO Con A blasts. (A), BlO.BR responder spleen cells treated with anti-Ia’ + C; (0) BlO.BR spleen cells treated with C alone.

in the A.TH anti-A.TL (anti-Iak) serum can be partially absorbed out by BlO.HTT or Bl0.A spleen cells but not by control A.TH spleen cells. (Table 3). BlO.HTT and Bl0.A share the I-E/C and I-A/B/J/E subregions with A.TL, respectively. This suggests that the regulatory cell probably expresses Ia determinants that are coded by the I-E/C and I-A/B/J/E subregions.

3x106 No.

of

regulatory

6~10~ cells

added

FIG. 2. The regulatory cells is Thy-l- and requires cell proliferation to suppress. BlO.BR spleen cells were treated with anti-Ia’ serum + C. 3 X IO6 of these treated cells were then cultures with 4 X lo6 irradiated C57BL/lO stimulator spleen cells and the indicated number of C-treated BlO.BR spleen cells (0) C + anti-thy-l treated BlO.BR spleen cells (0). or irradiated (2,000 R) BlO.BR spleen cells (A). After 4 days, the cultures were harvested and assayed for cytotoxicity against 2 X 10’ Wr-C57BL/ 10 Con A blasts. (A), cytotoxic activity for 3 X IO6 C + anti-Ia’-treated spleen cells; (0), cytotoxic activity for 3 X lo6 C-treated BlO.BR spleen cells; the cytotoxic activity of 6 X lo6 C-treated spleen cells against C57BL/lO target cells were 22.7 per culture. The anti-Thy-l + C-treated and irradiated BlO.BR spleen cells did not generate any detectable CTL activity against C57BL/lO.

88

TEH, TEH, AND YU TABLE 3 The Enhancing Effect of &a’ Serum is Absorbed by BIO.HTT and Bl0.A Spleen Cells”

Responder 4 4 4 4 4

x x x x x

Stimulator (2000 rad)

Treatment

IO6 BlO.BR IO6 BlO.BR lo6 BlO.BR lo6 BlO.BR IO6 BlO.BR

C C C C C

+ + + +

2 2 2 2 2

crIak (unabsorbed) LvIa’ (A.TH absorbed) oIak (BlO.HTT absorbed) oIa’ (B1O.A absorbed)

x x x x x

Lytic activity/culture (BlO target)

lo6 BlO lo6 BlO lo6 BlO IO6 BlO lo6 BlO

1.9 38.4 32.6

19.2 17.9

H-2 haplotypes Strain

K

I-A

I-B

I-J

I-E

I-C

S

G

D

BlO.BR A.TH A.TL BlO.HTT Bl0.A

k

k

k

k

k

k

k

k

k

f

t

f

it k k

t k d

i k d

f k d

k d d d d

S

S

L

S

S

k

t

’ The A.TH anti-A.TL serum was absorbed with appropriate normal spleen cells prior to use as follows: 1 ml of serum at l/l0 was absorbed twice for 1 hr at 0°C with 3-5 X 10’ spleen cells.

Anti-Ia serum specific only for the I-E/C subregions have similar properties as the anti-Ia serum specific for the entire I-region (Table 4). Here we showed that treating the responder population with anti-I-( EC)k + C also led to an enhancement of the CTL response to alloantigens and that the activity of this antiserum can be absorbed out by BlO.A(3R) spleen cells, which bear the K allele only at I-E. We TABLE 4 The Effect of B1O.S (7R) Anti-BlO.HTT (Anti-[-(E/C)‘) Cytotoxic Response to Alloantigens” Responder 4 x IO6 BlO.BR 4 X lo6 BlO.BR 4 x lo6 BlO.BR 4 x lo6 BlO.BR

Serum on

Stimulator (2000R)

Treatment C C + cyI-(E/C)k (unabsorbed) C + (YI-(E/C)’ (A.TH absorbed) C + C~I-(E/C)’ (Bl0.A (3R) absorbed)

2x 2x 2 x 2 x

Lytic activity/culture BlO Target

lo6 lo6 lo6 lo6

3.6 21.5 23.2 6.9

H-2 haplotype Strain

K

I-A

I-B

I-J

I-E

I-C

S

G

D

BlO.BR B1O.S (7R) BlO.HTT A.TH Bl0.A (3R)

k

k

k

k

k

k

k

k

S

S

S

S

S

S

s

S

f

;

k

i

;

s,

i

i

;

:

1

:

k d d d d

S

a Conditions used were similar to those described in Table 3. The cytotoxic indices obtained with unabsorbed, A.TH absorbed, or Bl0.A (3R) absorbed anti-I-(E/c)k serum were 44, 45, and 4% respectively.

REGULATION

OF CTL RESPONSES

89

BY Ia+ CELLS

conclude from these studies that the regulatory cell certainly expresses I-E/C determinants but we cannot rule out the expression of I-A/B/J-coded determinants on these cells. Regulation of CTL responses to TNP-modified self antigens by la+ cells. CTL responses to TNP-modified self-antigens are similarly affected by treating the responding population with anti-Iak or anti-I-( E/C)k + C (Table 5). Thus, pretreating the responder spleen cells with anti-Iak or anti-I-(E/C)k + C led to a 3.0- and 3.7fold increase in the response, respectively. On the other hand, pretreating with normal A.TH serum resulted in a response that was 1.3 times control value. We conclude that CTL responses to TNP-modified self-antigens are also regulated by an Ia+ cell. Estimation of the CLPfrequency in anti-la + C-treated responder cells. Culture systems capable of detecting single CLP to alloantigens have recently been reported (23-25). We have shown that culture conditions limiting for CLP can be obtained by culturing limiting numbers of responder cells with 1 X lo4 irradiated stimulator spleen cells and an optimum amount of costimulator (26), a lymphokine obtained by stimulation of spleen cells with Con A (27). This assay can be used to determine the frequency of CLP to alloantigens before and after anti-Ia treatment. One sees from Table 6 that the CLP frequencies to H-2d alloantigens in BlO.BR spleens which had been treated with anti-Iak + C were 1.1, 2.3, and 1.8 times that of the C-treated group in three different experiments. In these three experiments, treatment of control A.TH spleen cells with anti-Iak + C had no effect on the CLP frequencies to H-2d alloantigens when compared to the C control (data not shown). The approximately twofold increase in CLP frequencies to H-2d in BlO.BR spleen cells treated with anti-Iak + C is expected since the anti-Ia + C-treated cells should contain about twice as many CLP as the C-treated cells if CLP were Ia-. These data therefore confirm earlier conclusions that the increase in CTL response cannot be accounted for on the basis of an increase in the number of input CLP in the anti-Ia-treated population and argues in favor of the existence of an Ia+ regulatory cell. DISCUSSION Our studies confirmed earlier reports that T cells that respond in an MLR and CLP to alloantigens do not express Ia antigens that can be detected by conventional TABLE Effect of Anti-la

Serum on Cytotoxic

5

Responses to TNP-Modified

Self-Antigens” NcuT/culture

Responder

Treatment

4 X lo6 BlO.BR 4 X lo6 BlO.BR 4 x lo6 BlO.BR 4 X lo6 BlO.BR

C C + A.TH anti-A.TL serum C + a-[-(E/C)’ serum C + Normal A.TH serum

Stimulator (2000R) 2 2 2 2

x X X X

lo6 lo6 lo6 lo6

BlO.BR-TNP BlO.BR-TNP BlO.BR-TNP BlO.BR-TNP

BlO.BR <0.2 <0.2 <0.2 <0.2

BlO.BR-TNpb 2.10 6.40 7.80 2.70

’ Cultures were set up and assayed as described under Materials and Methods. b The Ncrt values of 2.10, 6.40, 7.80, and 2.70 correspond to percentage specific lysis values of 34, 72, 79, and 42% at effector to target ratios of 33, 21, 19, and 27:1, respectively.

+ + + +

aIa’ aIak cuIak cYIak

Expt 2

CLP/ 1O6cells 95% confidence limits X2

9.4 + 1.2 24.2 + 2.3

1.3 ++ 0.7 4.4 0.9

X2

0.8 k 0.7 0.9 + 0.4 4.6 k I.1 9.6 + I.2 CLP/ IO6 cells* 95% confidence limits

0.5 1.4 3.1 5.8

26.9 + 4.2 69.7 + 6.6

k f + +

1 x lo4 BlO.D2 I X IO4 BlO.D2

-0.5 6.2 27.8 63.8

2.8 0.6 7.3 + + 0.9

IO4 BlO.D2 lo4 BlO.D2 10.’ BlO.D2 IO4 BlO.D2

Expt I

11 X BlO.D2 X IO4 lo4 BlO.D2

1x 1X 1x I X

Stimulator spleen cells (2000 rad) f k + +

0.8 1.8 2.7 3.1

21.4 + 3.6 41.6 + 5.9

0.0 1.3 7.3 *+ 2.0

2.8 5.3 11.8 19.9

Expt 3 2112 4112 10112 12112 527 315 to 881

o/12 S/12 12112 12112 935 570 to 1533

2112 9112 12112 12112 1226 744 to 2020 1.53

2112 7112 12112 12112 980 598 to 1605 I .28

0.42

Expt 2

Expt I

Expt 3

812 495 to 1331 2.53

l/12 6112 12112 12112

0.59

l/12 5112 S/12 l2/12 444 266 to 738

No. of responding cultures

’ Cultures were set up in a volume of 0.20 ml in V-bottom microtiter trays as described under Materials and Methods. Responder cells were treated with A.TH anti-A.TL serum (l:lO) + C or C alone. Each culture received the indicated number of viable responder cells and the determined CLP frequencies to H-2d were those of the treated populations. Responding cultures were those whose counts exceeded the 95% confidence limits of the spontaneous control. Target cells = 2 x IO’ “CrP815. Spontaneous releases for the three experiments were 13, 7, and 13% of the maximum releasable counts, respectively. *Calculated according the method of Porter and Berry (20). All the data reported in this table are consistent with Poisson statistics.

3 X lo3 BlO.BR 1 X 10 4 BlO.BR

C C C C

C C C C

300 BlO.BR 1000 BlO.BR 3 x lo3 BlO.BR 1 x lo4 BlO.BR

300 BlO.BR 1000 BlO.BR

Treatment of responder cells

Responder spleen cells

Percentage specific lysis/culture (mean k SE)

Effect of Treatment of BlO.BR Spleen Cells with Anti-Ia’ + C on CLP Frequencies to Alloantigens”

TABLE 6

2

%

-g Lb

2 7 d

REGULATION

OF CTL RESPONSES

BY IaC CELLS

91

anti-Ia sera (7-9). Furthermore, our studies suggest that CTL responses to alloantigens and TNP-modified self-antigens are regulated by an Ia+ suppressor cell. This observation is in agreement with a recent report by Hayes et al. (12), where it was observed that CTL responses to minor histocompatibility antigens are also suppressed by an Ia+ cell. However, our findings are in disagreement with those reported by Plate (10) and Swierkosz et al. (4), where they reported that pretreating the responder cells with anti-Ia + C would eliminate Ia+ helper cells required for the induction of CTL responsesto alloantigens. It should be noted that the A.TH anti-A.TL serum used by Swierkosz et al. (4) was prepared against A.TL Con A blasts. We have also treated the responder population with anti-Iak serum raised against Con A blasts but obtained similiar results as with conventional anti-Ia” serum, i.e., we observed enhancement and not suppression of the cytotoxic response to alloantigens (data not shown). Since cytotoxic responsesin general are critically dependent on cell densities and the experiments reported by Swierkosz et al. (4) were done only at one cell dose it is conceivable that the suboptimal responses in the anti-Ia + C-treated group may be due to cell density effects. Nevertheless, it is clear from their studies that CLP are also Ia- since cytotoxic responses can be restored by the addition of bystander Fl T helper cells (4). The anti-Ia serum used by Plate was a Bl0.A anti-BlO.DZ antiserum ( 10). Thus, the effect observed by Plate may also be due to antibodies directed to H-2Kd which cross-reacted with C57BL/ 10 responder cells. The observation that CLP to TNP-modified self-antigens are also Ia- requires some comment. It has been reported by Cantor and Boyse (28) that CLP to TNPmodified self-antigens are Lyt-1+2+3+, in contrast to CLP to alloantigens, which are Lyt-l-2+3+. Thus, these two CLP may belong to two subclasses of cytotoxic cells. However, using a highly sensitive clonal assay for CLP, we were able to show that CLP to both alloantigens and TNP-modified self-antigens are Lyt-1+2+, suggesting that these two CLP cannot be functionally distinguished on the basis of their Lyt phenotype (29). The present study suggests that these two CLP also cannot be distinguished on the basis of their Ia phenotype. A great amount of experimental evidence has been recently gathered suggesting that CTL responsesare regulated by interleukin 2 (IL2) (30) a generic name given to a class of molecule(s) that play a fundamental role in the activation and subsequent clonal expansion of antigen-stimulated CLP (16, 27, 31, 32). The active moiety in costimulator that renders culture conditions limiting for CLP is IL2. It has also been shown that although macrophages are esential for IL2 production, it is produced by a Lyt-lf2- T cell (33, 34). It is not clear from our studies if the Ia+ cell regulates at the level of IL2 production. Studies are underway to determine the effect of removal of the Ia+ cell on the level of IL2 production in the MLR. The regulatory cell reported here appears to be Thy-l- and requires cell division before it can suppress. Thus, it differs from the I-J+ T suppressor cell reported in other systems (3, 35, 36). It also differs from the alloantigen activated T suppressor ceil that regulates MLR through soluble I-C region restricted factors (37). Sinclair et al. (38) have described an alloantigen-activated suppressor cell that appears to exert its suppressive effects primarily on the CLP. It is not clear if that cell and the regulatory cell reported here are related. It is conceivable that the regulatory cell may be a B cell or an immature macrophage since it is Thy-l-, Ia+, and requires cell division before it can suppress. If it is a B cell, then its removal may affect the

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production of alloantibody specific for the stimulating alloantigens or specific for the alloantigen receptor on CTL. Lalande et al. (32) has recently reported that alloantigen is only required during the first 12 hr of a CTL response. Consequently, alloantibody should have little effect on CTL responses during the later stages of the CTL response, assuming that most of the CTL were activated during the first 24 hr of the culture. If this is the case, regulation may be mediated by anti-idiotypic antibodies directed toward the CTL receptor. This possibility is currently being investigated. REFERENCES I. Press, J. L., Klinman, N. R., and McDevitt, H. O., J. Exp. Med. 144, 414, 1976. 2. Cowing, C., Schwartz, B. D., and Dickler, H. B., J. Immunol. 120, 378, 1978. 3. Murphy, D. B., Okumura, K., Herzenberg, L. A., and McDevitt, H. 0. Cold Spring Harbor Symp. Quant. Biol. 41, 497, 1976. 4. Swierkosz, J. E., Marrack, P., and Kappler, J. W., J. Exp. Med. 150, 1293, 1979. 5. Zinkernagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Streilein, J. W., and Klein, J., J. Exp. Med. 147, 897, 1978. 6. von Boehmer, H., Haas, W., and Jerne, N. K. Proc. Nut. Acad. Sci. USA 75, 2439, 1978. 7. Beverly, P. C. L., Woody, J., Dunkley, M., Feldmann, M., and McKenzie, I., Nature (London) 262, 495, 1976. 8. McKenzie, I. F. C., Pang, T., and Blanden, R. V., Immunol. Rev. 35, 181, 1977. 9. Lonai, P. In “Immune Recognition” (A. S. Rosenthal, Ed.), p. 683, Academic Press, New York. 10. Plate, J. M. D., Eur. J. Immunol. 6, 180, 1976. 11. Fujimoto, S., Matsuzawa, T., Nagakawa, K., and Tada, T. Cell. Immunol. 38, 378, 1978. 12. Hayes, C. E., MacPhail, S., and Bach, F. H., J. Exp. Med. 151, 1305, 1980. 13. Letarte, M., and Meghji, G., J. Immunol. 121, 1718, 1978. 14. Teh, H.-S., Letarte, M., Phillips, R. A., and Miller, R. G., Cell. Immunol. 37, 397, 1978. 15. Shearer, G. M., Eur. J. Immunol. 4, 527, 1974. 16. Symington, F. W., and Teh, H.-S., Stand. J. Immunol. 12, 1, 1980. 17. Watson, J., Aarden, L., Shaw, J., and Paetkau, V., J. Immunol. 122, 1633, 1979. 18. Shaw, J., Monticone, V., and Paetkau, V., J. Immunol. 120, 1967, 1978. 19. Miller, R. G., and Dunkley, M., Cell. Immunol. 14, 284, 1974. 20. Porter, E. H., and Berry, R. J., Brit. J. Cancer 17, 583, 1964. 21. Klein, J. In “Biology of the Mouse Histocompatibility-2 Complex,” p. 461. Springer-Verlag, New York. 22. Cantor, H., and Boyse, E. A., J. Exp. Med. 141, 1290, 1975. 23. Skinner, M. A., and Marbrook, J., J. Exp. Med. 143, 1562, 1976. 24. Teh, H.-S., Harley, E., Phillips, R. A., and Miller, R. G., J. Immunol. 118, 1049, 1977. 25. Lindahl, K. F., and Wilson, D. B., J. Exp. Med. 145, 508, 1977. 26. Paetkau, V., Miller, G., Gerhart, S., and Monticone, V., J. Immunol. 117, 1320, 1976. 27. Teh, H.-S., and Teh, S.-J., Nature (London) 285, 163, 1980. 28. Cantor, H., and Boyse, E. A., Cold Spring Harbor Symp. Quant. Biol. 41, 23, 1976. 29. Teh, H.-S., and Teh, S.-J., J. Immunol. 125, 1977, 1980. 30. Aarden, L., et al., J. Immunol. 123, 2928, 1979. 31. Larsson, E. L., and Continho, A., Nature (London) 280, 239, 1979. 32. Lalande, M. E., McCutcheon, M. J., and Miller, R. G., J. Exp. Med. 151, 12, 1980. 33. Shaw, J., Caplan, B., Paetkau, V., Pilarksi, L. M., Delovitch, T., and McKenzie, I. F. C. J. Immunol. 124, 2231, 1980. 34. Larsson, E. L., Iscove, N. N., and Continho, A., Nature (London) 283, 664, 1980. 35. Tada, T., Taniguchi, M., and David, C. S., J. Exp. Med. 144, 713, 1976. 36. Greene, M. I., Dorf, M. E., Pierres, M., and Benacerraf, B., Proc. Nut. Acad. Sci. USA 74, 5 118, 1977. 37. Rich, S. S., and Rich, R. R., J. Exp. Med. 143, 672, 1976. 38. Sinclair, N. R., Lees, R. K., Missiuna, P. C., and Vichos, E. E., Cell. Immunol. 27, 163, 1976.