'Journal of Immunological Methods, 19 (1978) 29--39

29

© Elsevier/North-Holland Biomedical Press

PHA-INDUCED CYTOTOXICITY OF HUMAN LYMPHOCYTES AGAINST ADHERENT HELA CELLS *

G. HUGES-LAW, G.C. DE GAST and T.H. THE

Clinical Immunology Unit, Department of Medicine, University of Groningen, The Netherlands (Received 21 April 1977, accepted 27 June 1977)

The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 pl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity. Supernatants of PHA-aetivated lymphocytes showed no cytotoxicity against adherent HeLa cells. Mitomycin treatment did not influence eytotoxic capacity. Removal of phagocytizing mononuclear cells reduced spontaneous cytotoxicity, but increased PHA-induced eytotoxicity. Adherent cells showed high spontaneous cytotoxieity, with little increase on addition of PHA. The method was evaluated for clinical applicability by testing mononuclear cells from 19 normal subjects and purified lymphocytes from 15 normal subjects. Purified lymphocytes showed a higher PHA-induced cytotoxicity with a smaller variation and greater ratio dependent increase in cytotoxieity than unseparated mononuclear cells. Results with fresh purified lymphocytes were reproducible.

INTRODUCTION

Lymphoid cells can be cytotoxic tO many other types of cell (Perlmann and Holm, 1969). This is one of the effector functions of cellular immunity, and constitutes an important defence mechanism. Cytotoxicity can be induced in vitro by stimulation of lymphoid cells; PHA is a good stimulant for in vitro assays of cytotoxic capacity (Holm et al., 1964). PHA causes aggregation of the lymphocytes to the target cells, followed by destruction of the latter. The results of cytotoxic assays are greatly influenced by the method used (Herberman and Oldham, 1975). Holm and Perlmann {1965, 1967a, b) studied lymphocyte cytotoxic capacity by 5'Cr release from Chang cells. Owing to the relatively high spontaneous release of 5,Cr, this assay is limited to short-term study, which may not be adequate for detection of this function (MacDonald et al., 1975). [3H]proline has been used as label of the * This study was supported by grants from Beecham B.V. and from the Stichting Koningin Wilhelmina Fonds, Nederlandse Organisatie voor de Kankerbestrijding.

30 target cells by Suciu-Foca et al. (1976). Reutilization of this label is difficult to control (Perlmann and Holm, 1969). We have evaluated the use of [3H]TdR, which is a stable marker, in an assay with adherent tumour cells. With [3H]TdR there is no spontaneous release and reutilization can be blocked by addition of excess cold thymidine (Perlmann and Holm, 1969). Thymidine release does not provide a sensitive method, because thymidine is released only when the nucleus of the target cells is also damaged. When detachment of adherent target cells was used as an indication of cell damage, [3H]TdR labeling proved to be suitable for measuring cytotoxic activity (Jagarlamoody et al., 1971). By this method we found a remarkable difference in cytotoxic activity between PHA stimulated and non stimulated lymphocytes after removal of the monocytes. MATERIALS AND METHODS

Effector cells Mononuclear cells were collected from heparinized blood from normal healthy volunteers by Ficoll--Isopaque gradient centrifugation according to the method of BSyum (1968). The isolated cells were suspended in medium RPMI 1640 (Gibco) with 25 mM HEPES, 100 U penicillin/ml, 100 pg streptomycin/ml and 10% pooled human inactivated (30 min at 56°C) serum. L y m p h o c y t e s were added in a ratio of 5 : 1, 10 : 1 and 20 : 1 (later 2.5 : 1 instead of 20 : 1) to the target cells (100,000, 200,000, 400,000 or 50,000 lymphocytes) with and without PHA. Purified lymphocytes were obtained by carbonyl iron treatment (type SF, GAF N.V. Delft, The Netherlands): 5 mg carbonyl iron per 1 ml blood incubated during 45 min at 37°C with repeated shaking and subsequently Ficoll--Isopaque gradient centrifugation. Purified monocytes were obtained by adherence to plastic. Mononuclear cells were incubated in plastic flasks (30 ml, Falcon, U.S.A.) in RPMI containing 20% human serum for 1 6 h at 37°C. Non-adherent cells were removed by decanting and careful washing once with prewarmed medium (37°C). The adherent cells were removed mechanically after incubation at 4°C for 1 h. Viability of the monocytes was good as judged by their phagocytosis of latex beads.

Target cells HeLa cells, an adherent cell line, were used as target cells. They were kept in continuous culture in MEM Eagle {Flow Lab., Scotland) with Earle's salts and 10% heat inactivated foetal calf serum. For the assay, HeLa cells were trypsinized (in 2% trypsin in Versene buffer for 2 min), washed and resuspended in MEM Eagle with heat-inactivated pooled human serum and counted. The viable cell number was never below 95% by trypan blue exclu-

31

sion. HeLa cells were seeded in flat-bottom microtiter plates {20,000 HeLa cells per well). At the same time 0.5 pCi [3H]thymidine (spec. act. 400 mCi/ mM) was added. The plates were incubated in a humidified atmosphere of 5% CO2 in air for 24 h to allow cell adhesion. The cells then grew into confluent monolayers. After 24 h each well was washed once using a biopipette. Excess cold thymidine was added in a volume of 0.025/well (25 /Jg/ml), to prevent reutilization of [3H]thymidine from dead HeLa cells.

Cytotoxic assay To each well 0.05 ml of the l y m p h o c y t e suspension with 10% heatinactivated pooled human serum and 0.05 ml medium, containing 1 pl PHA (stock solution) or 0.05 ml medium without PHA was added. Control wells without lymphocytes were included. The PHA stock solution, prepared by dissolving one vial of PHA (Phytohaemagglutinin, Burroughs Wellcome, England) in 5 ml medium, was kept at 4°C and used within one month. After 24 h incubation at 37°C in a humidified atmosphere of 5% CO2 in air, aggregates of cells could be seen in the wells with PHA with few normal looking HeLa cells. The detached target cells and lymphocytes were removed by washing with tapwater with an automatic cell culture harvester (Cryoson, Midden Beemster, Holland). Many of the less viable HeLa cells were removed in this way. The remaining adherent HeLa cells were trypsinized (2% trypsin in Versene buffer for 30 min at 37 ° C). The content of each well was harvested on a filter with the cell harvester. After drying for 1 h at 60°C, the filters were transferred to counting vials, 4 ml of scintillation fluid was added and counting performed in a liquid scintillation counter. The number of remaining target cells after contact with effector lymphocytes is indicated by the radioactivity (Jagarlamoody et al., 1971). Each experiment was done in triplicate, the medium control in 6 replicates. The cytotoxic activity was calculated using the medium control as base line, according to the formula: % cytotoxicity = 100-(dpm sample test/ dpm medium control × 100). Cytotoxic activity may also be expressed as increase in cytotoxicity between PHA stimulated and non stimulated lymphocytes. RESULTS

Labeling o f adherent target cells With 20,000 HeLa cells seeded in each well and cultured in the presence of [3H]thymidine for various periods, adequate thymidine labeling of the adherent cells was not reached until 16 h incubation (table 1, left part). After thymidine labeling, the labelled HeLa cells remained adherent for 24 h, b u t after this period spontaneous detachment occurred {table 1, right part). In all experiments thymidine content of adherent HeLa cells in the medium

32 TABLE 1 Target cell conditions. Thymidine labeling ( 1st step)

Assay (2nd step) after 24 h labeling

Time of incubation

Thym. content adh. HeLa cells (dpm)

Time of incubation

Thym. content adh. HeLa cells (dpm)

2h 4h 6b 8h 16 h 20 h 24 h

1,268 2,442 4,132 5,765 19,125 22,162 26,295

0 6 24 48 72

25,013 24,499 24,074 9,099 7,090

h h h h h

control varied between 20,000 and 30,000 dpm after 24 h labeling followed by 24 h incubation. Dose response curve PHA (fig. 1) PHA was tested in final concentrations of 100, 30, 10, 3 and 1 pl of the stock solution per ml culture medium at different lymphocyte-target cell ratios. In preliminary experiments PHA alone without lymphocytes showed no cytotoxic activity against adherent HeLa cells. Maximal cytotoxicity was obtained with a PHA dose of 10 pl/ml for 20 : 1 and 10 : 1 lymphocytetarget cell ratios but a 5 : 1 ratio gave somewhat higher cytotoxicity at a PHA dose of 3 pl/ml. Assays were subsequently done at a concentration of

°1, c y t o t o z i c i t y

IOO.

8o J

6oi

/

J

40 20 0

h .....0, pI4Jl, t00

PPIA ]0

PHA I0

PHA ~)

PHA )

~,rat,0b ~ P~A 0

PHA

conc "u~m L

Fig. 1. P H A dose response curve. The influence of various concentrations o f PHA on the cytotoxic capacity was tested. Points represent the mean o f 3 e x p e r i m e n t s w i t h mononuclear cells from different persons. A final concentration of 10 /JI P H A / m l appeared to

be optimal.

33 Day response

curve PFIA-it~duced

cytotolictty

% cytotoxictty I 1001

j'j:

80"

3B',.

60"

, ,."

'~0"

I

I ~1 .i.

Ii

/ 20"

/ . . . . .

0 r ~ LS"/" 6

i

h

18

72

time

(hrs)

Fig. 2. Effect o f i n c u b a t i o n time. P H A - i n d u c c d c y t o t o x i c i t y was s t u d i e d a f t e r various t i m e s o f i n c u b a t i o n . T h e results r e p r e s e n t m e a n o f 3 ratios with m o n o n u c l e a r cells from 2 donors. C y t o t o x i c i t y for each 24 h period is expressed as the p e r c e n t c y t o t o x i c i t y a f t e r s u b t r a c t i o n o f the c y t o t o x i c i t y in t h e p r e v i o u s period.

10 pl/ml PHA. Supernatants of lymphocytes (5 × 106 in 1 ml) incubated with PHA 10 pl/ml for 24 h showed no cytotoxicity against adherent HeLa cells.

Time o f incubation (fig. 2) PHA-induced cytotoxicity was assayed after 6, 24, 48 and 72 h incubation, at 3 lymphocyte-target cell ratios ( 2 0 : 1, 1 0 : 1 and 5 : 1 ) in two separate experiments with different lymphocytes. The medium control without lymphocytes showed the same number of adherent HeLa cells after 6 and 24 h incubation, as measured by their [3H]thymidine content, but a decline thereafter (see table 1). The percent cytotoxicity in the last 2 periods (second and third day) was therefore corrected for the spontaneous detachment of HeLa cells occurring in the medium control.

Influence o f mitomycin treatment To block DNA synthesis the mononuclear cells suspension was incubated with mitomycin (Sigma Chemical Co., 50 pg/ml, 30 min at 37°C) before the cytotoxic assay. Cells from four donors were tested. No consistent increase or decrease in spontaneous cytotoxicity was observed (table 2). None of the four donors showed a decline in PHA-induced cytotoxic capacity after mitomycin treatment. As shown in table 2, the mitomycin treatment effectively blocked DNA synthesis.

34 TABLE 2 Effect of m i t o m y c i n t r e a t m e n t ( m o n o n u c l e a r cells).

Exp. Exp. Exp. Exp.

1 2 3 4

Mean

Spontaneous cytotoxicity *

PHA-induced cytotoxicity *

PHA-induced DNA-synthesis **

be fore

after

be fore

after

before

after

65 44 32 16

71 66 33 --9

97 90 67 42

97 98 94 49

20,064 9,000 7,059

71 118 12.t

39

40

74

85

n.d.

* Expressed as p e r c e n t c y t o t o x i c i t y c o m p a r e d with the m e d i u m c o n t r o l . R a t i o of lymp h o c y t e s to target cells = 10 : 1. ** Expressed as t h y m i d i n e u p t a k e in d p m of 1 0 0 , 0 0 0 l y m p h o c y t e s c u l t u r e d for 3 days with PHA 10 p l / m l in m i c r o t i t e r plates.

Removal of phagocytic cells Phagocytic cells were removed by incubation with carbonyl iron powder. After this procedure monocyte contamination ranged from 2--4%. In table 3 results with lymphocytes from 4 different donors are shown. After removal of phagocytizing cells a higher PHA-induced cytotoxicity (85% after removal against 62% before), but a lower spontaneous cytotoxicity (8% after removal against 26% before) was found.

TABLE 3 I n f l u e n c e o f removal of p h a g o c y t i z i n g cells. % monocytes

Exp. Exp. Exp. Exp. Mean

1 2 3 4

Spontaneous cytotoxicity *

PHA-induced cytotoxicity *

before

after

before

after

before

after

24 15 12 13

3 4 2 2

42 38 16 8

2 21 3 7

90 72 50 36

91 87 88 75

16

3

26

8

62

85

* Expressed as p e r c e n t c y t o t o x i c i t y c o m p a r e d to the m e d i u m c o n t r o l . Ratio of l y m p h o cytes to tarRet cells = 10 : 1.

35 TABLE 4 Influence of monocyte depletion and enrichment by adherence to plastic on spontaneous and PHA-induced cytotoxicity. Donor A Spontaneous 5 :1* 10:1 PHA-induced 5 :1 10 : 1 Donor B Spontaneous 5:1 10 : 1 PHA-induced

Unseparated (81% ly, 19% mono)

Non-adherent (92% ly, 8% mono)

Adherent (14% ly, 86% mono)

12 6

31 63

21 (17) 61 (59)

32 (20) 69 (63)

49 (18) 77 (14)

Unseparated (86% ly, 14% mono)

Non-adherent (96% ly, 4% mono)

Adherent (41% ly, 49% mono)

4 ** 2

8 5

5 3

20 44

5 :1

52 (44)

74 (69)

40 (20)

10 : 1

75 (70)

90 (87)

74 (30)

Lymphocyte-target cell ratio. ** Expressed as percent cytotoxicity compared to medium control; in parentheses the difference between PHA-induced and spontaneous cytotoxicity at each ratio. *

Effect o f adherent mononuclear cells In 2 e x p e r i m e n t s s p o n t a n e o u s and P H A - i n d u c e d c y t o t o x i c i t y o f plastic a d h e r e n t cells ( m o n o c y t e enriched) were c o m p a r e d with t h o s e o f nona d h e r e n t cells and those o f u n s e p a r a t e d m o n o n u c l e a r cells (table 4). The a d h e r e n t cells s h o w e d higher s p o n t a n e o u s c y t o t o x i c i t y whereas little increase in c y t o t o x i c i t y was observed on a d d i t i o n o f PHA. In c o n t r a s t the n o n - a d h e r e n t cells s h o w e d low s p o n t a n e o u s c y t o t o x i c i t y and an evident increase in c y t o t o x i c i t y o n a d d i t i o n o f PHA.

Results with mononuclear cells and purified lymphocytes from normal donors To evaluate the m e t h o d , m o n o n u c l e a r cells f r o m 19 n o r m a l d o n o r s , in age varying f r o m 2 0 - - 6 0 years, and purified l y m p h o c y t e s f r o m 15 o t h e r n o r m a l d o n o r s in the same age range, were tested. T h e results for s p o n t a n e o u s and P H A - i n d u c e d c y t o t o x i c i t y are s h o w n in fig. 3. S p o n t a n e o u s c y t o t o x i c i t y was evident ( m e a n ~_ S.D. 21% -+ 18} at a l y m p h o c y t e - t a r g e t cell ratio o f 20 : 1. P H A i n d u c e d a significantly higher c y t o t o x i c i t y which was c o r r e l a t e d with the l y m p h o c y t e - t a r g e t cell ratio (mean +-S.D. 46% _+ 26, 61%-+ 25 and 70% -+ 20 at ratios o f 5 : 1, 10 : 1 and 20 : 1 respectively). T h e results with

36 P H A _ i n d u c e d cytotoxicity in controls ( mononuctear

celts )

% c ytotoxicity spont@neou5

I00,

PHA-[ndu¢¢d

8o 6oi

~

4oi 2oi

A 4-T

|

i I|

ol

•

: " *

:

:

T I

Z

ratio

u $

.5,

5 1

101

201

mean

5,1

10 1

201

rne~n

Fig. 3. S p o n t a n e o u s and PHA-induced c y t o t o x i c i t y with mononue]ear cells from 19 normal persons. PHA-indu(.ed

cytotoxlcity

( purifi,d °/o

*n

controls

lymphocyt¢$)

cytotoxlcity

5pcntareous

P~A

.~duce~

iO0-

BO

I

60 |

le

4O • o~_-

g"

t

20,

0

,.:

i _.~_

$

i

N

-r

•

V

=

;

rQtlO

t

T

|

$

-20,

751

S '.

I(] 1

mean

2~ ~

51

10 *

mean

Fig. 4. Spontaneous and PHA-induced cytotoxicity with purified lymphocytes from 15 normal persons. Monocytes were removed from the mononuclear cell suspension by carbonyl iron phagocytosis before the Ficoll--Isopaque gradient centrifugation. With purified lymphocytes lower ratios can be used than with mononuclear cells (lymphoeytes and monocytes).

37 p u r i f i e d l y m p h o c y t e s ( m o n o c y t e s r e m o v e d b y c a r b o n y l i r o n ) f r o m 15 o t h e r d o n o r s are s h o w n in fig. 4. V e r y l i t t l e s p o n t a n e o u s c y t o t o x i c i t y w a s o b s e r v e d ( 3 % , 5% a n d 8% a t r a t i o s o f 2 . 5 : 1, 5 : 1 a n d 1 0 : 1). N e a r l y t h e s a m e P H A - i n d u c e d c y t o t o x i c i t y as w i t h m o n o n u c l e a r cel l s w as r e a c h e d a t l o w e r l y m p h o c y t e - t a r g e t cell r a t i o s ( m e a n -+ S . D . 3 5 % -+ 17, 5 7 % + 2 2 a n d 8 1 % +_ 17 at r a t i o s o f 2 . 5 : 1, 5 : 1 a n d 10 : 1). T h e v a r i a t i o n w a s s m a l l e r a n d t h e r a t i o d e p e n d e n t i n c r e a s e in c y t o t o x i c i t y g r e a t e r .

TABLE 5 Reproducibility of the cytotoxicity test against adherent HeLa cells. Donor

Source of cell suspension

PHA-induced

Spontaneous 5:1

10:1

20:1

mean

5:1

10:1

20:1

mean

A

Fresh mononuclear cells

5 9 1 7 5

2 4 --3 12 16

17 13 5 12 12

8 9 1 10 11

45 47 42 33 37

55 74 59 52 59

78 90 70 61 76

59 70 57 49 57

B

Frozen stored mononuclear cells

3 6

3 13

3 3

3 7

59 39

89 62

98 88

82 63

C

Frozen stored mononuclear cells

12 6

10 6

10 16

11 9

52 53

74 89

96 95

74 79

D

Frozen stored mononuclear cells

11 2 5 --8

14 6 1 --13

18 0 -1 --5

14 3 2 --9

62 61 38 28

88 84 71 63

95 96 90 84

82 80 66 58

mean

2.5:1

5:1

10:1

mean

2.5:1

5:1

10:1

E

Fresh purified lymphocytes

--3 --5 5

--2 --10 --13

1 2 --9

--1 --4 --6

89 94 93

98 97 92

94 98 87

94 96 91

F

Fresh purified lymphocytes

--2 7

6 14

14 14

6 12

18 17

32 38

72 65

41 40

G

Fresh purified lymphocytes

--1 --6

--7 --4

--6 --5

--5 --5

51 42

72 83

91 98

71 74

H

Fresh purified lymphocytes

--10 --2

--14 7

--9 --11

--11 --2

36 29

58 67

89 92

61 63

I

Fresh purified lymphocytes

--4 --1

4 6

--10 6

--3 4

25 19

31 23

56 43

37 28

J

Fresh purified lymphocytes

--5 3

--9 1

--6 --1

--7 1

10 19

23 35

55 55

29 36

K

Fresh purified lymphocytes

6 7

6 --4

1 3

4 2

30 16

46 31

50 37

.12 35

38

Reproducibility o f the method To test reproducibility cytotoxicity tests were repeated at weekly intervals with fresh mononuclear cells and purified lymphocytes from several normal persons and patients {table 5). Less fluctuation in cytotoxicity occurred with fresh mononuclear cells than with frozen stored mononuclear cells taken on different days and tested at the same time. Having demonstrated the need to deplete the cells of monocytes for proper evaluation of lymphocyte cytotoxicity, we also tested purified lymphocyte suspensions. Table 5 shows that results with fresh purified lymphocytes from controls as well as from patients were reproducible. DISCUSSION This method, in which adherent human turnout-derived target cells (HeLa cells) are used, provides a suitable measure of the cytotoxic capacity of human lymphocytes. HeLa, which is a rapidly growing established cell line derived from cervix carcinoma adapted to tissue culture conditions, is a readily available source of adherent target cells. Thymidine labeling of the target cells, which is easy to perform and quite consistent, abolishes the necessity for optical counting which remains subjective. The viable adherent target cells left at the end of the assay are harvested automatically and their thymidine is content counted. Supernatants of PHA-activated lymphocytes showed no cytotoxic activity against adherent HeLa cells. The optimal PHA concentration (10 pl/ml) is approximately the same as that used by Holm and Perlmann (1967b) in a SlCr release assay. In their studies PHA-induced cytotoxicity was proportional to blast transformation, but we did not find a definite increase in cytotoxicity after an incubation time of more than 24 h, although this was difficult to evaluate owing to spontaneous detachment of target cells in the absence of lymphocytes. PHAinduced cytotoxicity against HeLa cells appeared to be independent of DNA synthesis, however, since mitomycin treated cells showed no decrease in cytotoxic capacity. This is in agreement with the findings of Dawkins and Zilko {1975). Phagocytic mononuclear cells have an important effect on results in this cytotoxicity test against adherent t u m o u r cells, since their removal decreased spontaneous cytotoxicity and increased PHA-induced cytotoxicity. Monocytes enriched by adherence to plastic showed a high spontaneous cytotoxicity, very little increased by addition of PHA. Contradictory results on this point have been reported: De Bracco et al. {1976), using the ~Cr release assay on chicken erythrocytes, found that iron treatment abolished PHA-induced cytotoxicity, while Fauci et al. (1976), using the same method, found no effect of monocyte depletion by nylon wool filtration. It may be relevant that in our test adherent turnout-derived target cells are used. From our results it is clear that mononuclear phagocytes must be removed to measure

39

PHA°induced lymphocyte cytotoxicity on adherent HeLa cells in order to avoid high spontaneous cytotoxicity and interference with PHA-induced cytotoxicity. With purified lymphocytes, after removal of phagocytic cells, lower ratios of lymphocytes to target cells (2.5 : 1, 5 : 1 and 10 : 1) were needed to give definite PHA-induced cytotoxicity, which was proportional to the ratio used. These ratios are much lower than those (up to 50 : 1) used by Holm and Perlmann {1967}, Stejskal et al. (1973a, b) and by Suciu-Foca et al. (1976}. Thus our method is more sensitive and has in addition the great advantage that only a small amount of blood is required (3.2 × 106 lymphocytes per donor). Using purified lymphocytes we found in our control group hardly any spontaneous cytotoxicity by the lymphocytes and the results were reproducible. So far little is known about the mechanism of PHA-induced cytotoxicity. Viable effector cells and contact between effector and target cell are absolute requirements (Holm and Perlmann, 1967b; Asherson et al., 1973). Cytotoxicity is more pronounced when effector and target cell are different with regard to histocompatibility antigens, suggesting an immunological recognition step (Holm and Perlmann, 1967a). This finding was confirmed by another study using human and mouse lymphocytes as effector cells, stimulated by different agents (Stejskal et al., 1973b). Thus PHA-induced cytotoxicity against adherent HeLa cells is a quick, easy and reproducible method of studying the cytotoxic capacity of human lymphocytes and requires only a small number of lymphocytes. Removal of monocytes is necessary for an adequate evaluation of this lymphocyte effector cell function. REFERENCES Asherson, G.L., J. Ferluga and G. Janossy, 1973, Clin. Exp. Immunol. 15,573. B6yum, A., 1968, Scand. J. Clin. Lab. Invest. 21, suppl. 97, 77. De Bracco, M.M.D., M.A. Isturiz and J.A. Manni, 1976, Immunology 30, 325. Dawkins, R.L. and P.J. Zilko, 1975, Nature 254, 144. Fauci, A.S., J.E. Balow and K.R. Pratt, 1976, J. Clin. Invest. 57,826. Herberman, R.B. and R.K. Oldham, 1975, J. Natl. Cancer Inst. 55, 749. Holm, G., P. Perlmann and B. Werner, 1964, Nature 203,841. Holm, G. and P. Perlmann, 1965, Nature 207,818. Holm, G. and P. Perlmann, 1967a, Immunology 12, 525. Holm, G. and P. Perlmann, 1967b, J. Exp. Med. 125,721. Holm, G., C. Franksson, A.C. Campbell and I.C.M. MacLennan, 197:t, Clin. Exp. Immunol. 17,361. Jagarlamoody, S.M., J.C. Aust, R.H. Tew and C.F. McKhann, 1971, Proc. Natl. Acad. Sci. U.S.A. 68, 1346. MacDonald, H.R., B. Sordat, J.C. Cerottini and K.T. Brunner, 1975, J. Exp. Med. 142, 622. Perlmann, P. and G. Holm, 1969, Adv. Immunol. 11, 117. Stejskal, V., S. Lindberg, G. Holm and P. Perlmann, 1973a, Cell. Immunol. 8, 82. Stejskal, V., G. Holm and P. Perlmann, 1973b, Cell. Immunol. 8, 71. Suciu-Foca, N., J.A. Buda, F. Herter, A. Molinaro, J. Broell and K. Reemtsma, 1976, J. Surg. Oncol. 8, 75.