Tissiie Ant igens (1975), 6, 279-285

Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

The Use of Directly Frozen Cells in LDA Assay

CHRISTIANE WERNER, FERNANDO BOTELLA, MOIRA EUGSTER, CLAUDE LEGENDRE AND MICHEL JEANNET

Transplantation Immunology Unit, Division of Immunology and Allergy, HBpital Cantonal,

Geneva, Switzerland

A method of direct freezing is described which requires a cryoprotective medium consisting of human AB serum and DMSO, an ultra-low temperature freezer and a liquid-nitrogen re- frigerator. Fresh and directly frozen cells were compared in the LDA assay. Direct freezing is quite reliable for normal lymphocytes, PHA transformed lymphocytes or leukaemic blasts used as targets. However, a controlled freezing procedure may be necessary to preserve the effector cell activity.

Received for publication 17 January, accepted I July 1975

Lymphoid cells from non-sensitized indi- viduals have the capacity to lyse IgG anti- body-coated target cells in uitro in the ab- sence of complement (Perlmann et ai. 1972, Hersey et al. 1973, Cerottini & Brunner 1974). This cytotoxic mechanism has been described in kidney transplants (Jeannet et al. 1975, d’Apice & Morris 1974), parous women ( McConnachie & Dossetor 1973), multiple transfused pa- tients (Yust et al. 1974) and leukemic pa- tients (Hersey et al. 1973, Jeannet et al. 1974). In this assay, the use of frozen cells as targets or effectors makes it possible to study the immune reactivity of patients over a long period of time. Two main methods of cryopreservation have been

described using a controlled programmed freezer (Powles et al. 1973, Guttermann et al. 1973, Thomson & O’Connor 1971, Eijsvoogel et al. 1973) or freezing directly in an ultra-low temperature freezer (Wood et al. 1972, Stopford et al. 1972).

Our experiments were designed to study the possibility of using directly frozen cells stored in liquid nitrogen in the LDA assay without loss of their immunological pro- Ferties.

Materials and M e t h o d s 1. Freezing and T h a w i n g of the Cells Lymphocytes were obtained by Ficoll-Iso- paque flotation. The cells were washed

This work was supported by grant no. FOR.062.AK.74 ( 3 ) from the Ligue Suisse Contre le Cancer and grant no. 3.8580.72 from the Fonds National Suisse de la Recherche Scientifique.

280 WERNER ET AL.

twice with Hank's balanced salt solution (HBSS) and suspended in inactivated hu- man AB serum at a concentration of 10- 20X 106Jm1. One ml of the suspension was dispensed in a 2 ml polypropylene am- poule (238JS 2 ml Dynatech) placed on ice. An equal volume of freezing mixture consisting of 20 "/o dimethylsulfoxide (DMSO) and 80 "/. AB serum was added drop by drop. Ampoules were then placed on a rack in an ultra-low temperature freezer at - 70" C for at least 30 min before being stored in a liquid nitrogen refrigerator.

When required, ampoules were thawed rapidly in a water-bath at 37" C. Ten vol of Eagle's minium essential medium (MEM) with 10 % AB serum were added very slowly to one vol of cells. The cells were counted in a haematocytometer chamber and viability was determined by trypan blue dye exclusion.

In order to evaluate the usefulness of this freezing method, some cells were also frozen at a controlled rate of 1OCJmin using a programmed freezer (Planer Ltd. R 201). For the LDA assay, target cells were frozen after 3 days of culture in pla- stic bottles in the presence of PHA, final dil. 1 : 800 (Phytohaemagglutinin M, Dif- co). Leukemic blasts which were to be used as targets were frozen after separa- tion. Frozen lymphocytes were also used in a few experiments as effector cells.

2. Lymphocyte-Dependent Cytotoxic

The LDA assay was performed according to Trinchieri et al. (1973) with some mo- difications. In brief, targets were thawed, washed once, suspended in 0.1-0.2 ml Tris buffer (pH 7.2) and labelled with 100 pCi of Cr51 (0.1 ml spec. act. 100- 500 mCi/mg Cr) ; the mixture was incu- bated for 1 h in a water bath at 37O C with frequent shaking. The cells were then

Antibody Assay (LDA)

washed three times and resuspended at a concentration of 0.2X 106 celldm1 in MEM with 10-20 % AB serum.

Effector cells were separated by Ficoll- Isopaque flotation, washed twice and re- suspended at a concentration of 2 X lO6J ml. The LDA assay was performed in quadriplicate using round-bottom micro- plates. Sera to be tested were inactivated and diluted in MEM with 10-20 % AB serum. Fifty p1 of serum or plasma dilu- tion (usually 1 : 10) were mixed with 50 p l of target cells ( 1 X 104Jwell) suspended in MEM-Hepes; after a f / z h incubation at 4' C, 100 pl (2X l@/well) effector cells were added. The ratio target: effec- tor was therefore 1 : 20.

The following controls were included:

a) maximum release ( M ) : substitution of serum and effector cells by 150 p1 of saponin 10 % (Zaponin, Coulter Elec- tronics).

b) spontaneous release (S) : substitution of serum by 50 pl of MEM with 20 o/a AB serum.

c) Crsl incorporation of each target pop- ulation.

After a 4 h incubation at 37" C, the plates were centrifuged at 900 g for 10 min at 4 O C and the supernatants (100 p l ) removed from each well and counted for Cr61 release. Percentage of specific lysis (cytotoxicity) is calculated according to the formula (E-S)J (M-S) X 100, where E is the experimental release, S the spontaneous release and M the maximum release. A positive test is defined by a per- centage of specific lysis greater than 4 % and an experimental release significantly greater ( P < 0.05) than the spontaneous release, using Student's t test.

Results Preliminary experiments were carried out

DIRECT FREEZING METHOD

Cr51 incor- poration

cpm * SD

28 1

Maximum Spontaneous

(saponin) cpm -t SD P P release release

cpm i SD

CR5’ LABELLING OF FRESH,CONTROLLED FROZEN AND DIRECTLY FROZEN TARGET CELLS

0 fresh targets 1 CR5’ incorporated controlled frozen targets 2 maximum release (saponin 10 %) directly frozen targets 3 spontaneous release

m 0 -

0 :i 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

target : HL-A 1,9,W17,W21 HL-A 2,W32,7

Figure 1 . Comparison of Cr51 labelling of fresh, controlled frozen and di- rectly frozen cells, Normal lymphocytes were transformed into PHA blasts after thawing. Maximum release was obtained by saponin-mediated lysis and spontaneous release was determined by Cral activity in the super- natant. The “bars” represent the standard deviation.

Target cells

Fresh Controlled frozen

Directly frozen

P

< 0.05b

N.SC <0.01b

a % of maximum release.

C Statistical significance between controlled frozen and directly frozen cells. Statistical significance between fresh and frozen cells.

in order to compare (21-51 labelling of con- both methods of freezing. Cr51 incorpora- trolled frozen and directly frozen normal tion does not vary greatly between the two lymphocytes (Fig. 1) . After thawing, the methods, but is usually slightly better viability determined by trypan blue dye using fresh cells. exclusion varied between 85-100 % and Table 1 shows an experiment carried the loss of cells was usually 2 M O ”/. with out with PHA transformed lymphocytes

282

Target cellsC Serum taI release Iysis HL-A

WERNER ET AL.

Pb

- Exp. no.

- 1.

2.

3.

4.

CrS1 incor- Maximum Spontaneous Directly frozen poration release release

cpm +- SD cpm ir SD cpm c SD target cells

Table 2 Comparison of fresh and directly frozen target cells (PHA transformed lymphocytes)

in the L D A assay using monospecific anti-HL-A sera

~~

Experi- Specific mentalb lysis release

cpm k SD

1

2, X, W5, W10 Fresh

9, X, 12, W18 Fresh

2, 28, 12, W21 Fresh

idem Fresh

idem Fresh

idem Fresh

1, 9, 8, W5 Fresh

idem Fresh

idem Fresh

Frozen

Frozen

Frozen

Frozen

Frozen

Frozen

Frozen

Frozen

Frozen

Anti-HL--A2 511 i- 31 (220/,)a 740 I- 59 (35%)

Anti-HL--Al2 480 C 52 (31%) 684 -i- 46 (400/0)

Anti-HL-A2 588 i- 51 (26%) 488 3: 36 (37%)

Anti-HL--A28 588 i 51 (26%) 488 i- 36 (37%)

Anti-HL--A12 588 +- 51 (26%) 488 I- 36 (37%)

Anti-W21 588 -t 51 (26%) 488 t 36 (37%)

Anti-HL--A8 514 t 34 (21 yo) 604 i- 26 (25%)

Anti-HL--A1 514 t 34 (21%) 604 i 26 (25%)

Anti-W5 514 t 34 (21 yo) 604 i- 26 (25%)

857 t 7 7 19 996 t 82 18 826 * 22 33

1008 C 4 4 34 940 * 24 21 672 -t 51 22

1086 C 52 31 721 i- 41 28 852%32 16 6 3 0 i 3 3 17

1006 t 31 29 667 k 3 6 21

1802 i-66 68 1248 k 3 6 37 1424i-60 48 1160 t 6 0 32 832 1 7 2 16 902 * 84 17

<0.001 < 0.001 <0.001 <0.001 <0.001 <0.001 <0.001 < 0.001 <0.001 < 0.001 <0.001 t0.001 <0.001 <0.001 < 0.001 <0.001 < 0.001 <0.001

a % of maximum release. b Statistical significance between experimental and spontaneous releases. C Control values (cpm -t SD):

Exp. no. 1 fresh targets: CrS1 incorp.: 2454 t 79, max. rel.: 2245 * 177 froz. targets: ,, ,, : 2036 i- 17, ,, ,, : 2098 _C 59

Exp. no. 2 fresh targets: ,, ,, : 1708 t 16, ,, ,, : 1508 C 52 froz. targets: ,, ,, : 2053 i 162, ,, ,, : 1684 t 92

Exp. no. 3 fresh targets: ,, ,, : 2479 5 212, ,, ,, : 2191 -t- 111 froz. targets: ,, ,, : 1459 f 64, ,, ,, : 1318 i- 71

Exp. no. 4 fresh targets: ,, ,, : 2393 rt 25, ,, ,, : 2390 C 132 froz. targets: ,, I , . . _ _ _ ,, ,, : 2324 C 116

Table 3 Directly frozen leukaemic blasts compared with directly frozen normal PHA blasts in the LDA assay

~ ' " ' I PHA-normal blasts 4401 * 147 3859 i 609 1407 1 89 (36%)a 2841 * 298 58 <0.001 Leukaemic blasts (A.S.) 2238 i- 105 2060 -i; 57 663 2 62 (32oj,) 1132 t 83 33 <0.001 Leukaemic blasts (L.M.) 1828 i- 91 1699 1 184 484 -+ 27 (280/6) 1304 115 67 <0.001

a % of maximum release. Using a multispecific anti-HL-A serum. Statistical significance between experimental and spontaneous releases.

DIRECT FREEZING METHOD 283

C.YTOTOXIC ACTIVITY OF F R E S H A N D DIRECTLY F R O Z E N EFFECTOR CELLS

0 f resh effector c e l l s f rozen effector ce l ls

target HL-A 2,W25,12, W15

serum N O : 1 2 1 2 e f fec tor : HL-A 3,1tJ7,W1O HL-A 2 ,W5,W10

Figure 2. Cytotoxic activity of fresh and directly frozen effector cells. Fresh PHA transformed lymphocytes were used as targets. Experiments were per- formed with multispecific anti-HL-A sera.

intended to be used as targets in the LDA assay. The incorporation of Cr51 in cells frozen by the two methods is significantly lower than in fresh cells, but still quite sufficient; consequently, maximum release and spontaneous release are lower. Dif- ferences between the two methods of freezing are not significant. Fresh and di- rectly frozen cells used as targets were compared in the LDA assay. Some experi- ments are shown in Table 2. The ratio of spontaneous releasejmaximum release is usually higher in frozen cells than in fresh cells. The specific lysis obtained with mono- specific anti-HL-A sera is usually com- parable between fresh and frozen targets. In some cases (exp. nos. 3 and 4) specific lysis is clearly higher with fresh targets. Directly frozen blast cells from two pa- tients with acute myeloblastic leukaemia were used as targets in many experiments; a typical experiment (Table 3 ) shows that Cr51 incorporation is lower with frozen

leukaemic blasts than with frozen PHA blasts. However, the specific lysis was usually comparable to that obtained with PHA blasts.

Directly frozen effector cells were tested in a few experiments (Fig. 2 ) . The specific lysis was found to be always lower with frozen cells than with fresh cells.

Discussion I t has been shown that HL-A typing can be Ferformed with accuracy on carefully frozen cells without using a controlled programmed freezer (Stopford et al. 1972, Wood et al. 1972). I n this study, directly frozen cells have been tested in compari- son with fresh cells in the LDA assay. Di- rect freezing requires a cryoprotective me- dium consisting of AB serum and DMSO, an ultra-low temperature freezer and a liquid nitrogen container. I t might per- haps be possible to avoid the stage in the

284 WERNER ET AL.

ultra-freezer by using a special biological freezer (BF-6) designed for freezing small quantities of biological materials, but we have not tested this system. Our experi- ments suggest that the use of an expensive and time-consuming programmed freezer is not necessary for freezing normal lym- phocytes, PHA blasts or leukaemic blasts used as targets in the LDA assay, since di- rect freezing seems to be a reliable rneth- od. The experiments performed with mo- nospecific anti-HL-A sera show that the same HL-A antigens can be detected on thawed frozen cells as well as on fresh cells (Table 2 ) . The use of frozen targets has considerably facilitated the perforrn- ance of LDA tests involving donors with special HL-A types, leukaemic patients and cadaver kidney donors. Preliminary experiments show that this method of di- rect freezing is less adequate in preserving the immunological properties of effector cells; a controlled freezing of normal lym- phocytes used as effector cells in the LDA assay may be necessary in order to get a maximally efficient population of K cells. In our experience, however, the use of a pool of effector cells from three random blood donors obviates the need for selec- tion of special HL-A types and therefore renders freezing of effectors less manda- tory.

References d’Apice, J. F. & Morris, P. J. (1974) The role

of antibody-dependent cell-mediated cytoto- xicity in renal allograft rejection. Transplan- tation 18, 20-30.

Cerottini, J. C. & Brunner, K. T. (1974) Cell- mediated cytotoxicity, allograft rejection and tumor immunity. Adv. Immunol. 18, 67-132.

Eijsvoogel, V. P., du Bois,, M. J. G. J., van den Wal, R., Huismans, D. R. & Raat-Koning, L. ( 1973) Cryopreserved lymphocytes: function- al properties in vitro. Cryopreservation of Normal and Neoplastic Cells, ed. Weiner, R. S., Oldham, R. K. & Schwarzenberg, L., p. 101-106. Colloque de 1’I.N.S.E.R.M.

Guttemann, J. U., Mavligit, G. M., .McCredie,

K. B., Freireich, E. J. & Hersh, E. M. (1973) Preservation of leukemic cells for use in stud- ies of autologous immunity to leukemia anti- gens. Cryopreservation of Normal and Neo- plastic Cells, ed. Weiner, R. S., Oldham, R. K. & Schwarzenberg, L., p. 171-178. Colloque de 1’I.N.S.E.R.M.

Hersey, P., Cullen, P. & MacLennan, I. C. M. ( 1973) Lymphocyte dependent cytotoxic antibody activity against human transplanta- tion antigens. Transplantation 17, 9-16.

Hersey, P., MacLennan, I. C. M., Campbell, A. C., Harris, R. & Freeman, G. B. (1973) Cytotoxicity against human leukaemic cells. Clin. exp. Immunol. 14, 159-166.

Jeannet, M., Vassalli, P., Magnin, C. & Cru- chaud, A. (1974) Evaluation d‘une nouvelle mkthode de detection d’alloanticorps anti- lymphocytaires et d’anticorps anti-leucCmi- ques sptcifiques. Schweit. med. Wschr. 104, 1388-1389.

Jeannet, M., Vassalli, P. & Botella, F. (1975) Lymphocyte-dependent cytotoxic antibody in kidney transplantation. Transplant. Proc. (in press).

McConnachie, P. R. & Dossetor, J. B. (1973) Antibody-mediated cell-dependent immunity in human lymphocytes. Tissue Antigens 3, 303-308.

Perlmann, P., Perlmann, H. & Wigzell, H. ( 1972) Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells. Trans- plant Rev. 13, 67-90.

Powles, R. L., Balchin, L. A., Smith, C. & Grant, C. K. (1973) Some properties of cryo- preserved acute leukemia cells. Cryobiology

Stopford, C. R., MacQueen, J. M., Amos, D. B. & Ward, F. E. (1972) Some variations in lymphocyte freezing methods which do not affect cell viability. Tissue Antigens 2, 20-26.

Thomson, A. E. R. & O’Connor, T. W. E. (1971) Observations on cryopreservation of lymphocytes in chronic lyrnphocytic leukae- mia and normal human lymphocytes, Scand. J. Haematol. 8, 425438.

Trinchieri, G., de Marchi, M., Mayr, W., Savi, M. & Ceppellini, R. (1973) Lymphocyte antibody lyrnphocytic interaction (LALI) with special emphasis on H G A . Transplant. Proc. 5, 1631-1646.

Wood, N., Bashir, H., Greally, J., Amos, D. B. 6r Yunis, E. J. (1972) A simple method of freezing and storing live lymphocytes. Tissue Antigens 2, 27-31.

10, 282-289.

DIRECT FREEZING METHOD 285

Yust, I., Wunderlich, J. R., Mann, D. L. & Terry, W. D. (1974) Identification of lym- Christiane Werner phocyte-dependent antibody in sera from Unit6 d’immunologie de transplantation multiple transfused patients. Transplantation 18, 99-107.

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