TissueAntigens(1979), 13,189-194 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the authork)

Hemolytic Plaque Formation to Allogeneic Human Type A and B Red Cells' Mutsuo Sasaki and Paul I. Terasaki Department of Surgery, UCLA School of Medicine University of California, Los Angeles, California, U.S.A. Plaque-forming cells can be generated from peripheral blood lymphocytes provided that the lymphocytes are prestimulated by in vitro sensitization in the presence of pokeweed rnitogen. Of 91 blood type 0 persons whose lymphocytes were stimulated with red cell A antigen, 52% responded with plaque formation, whereas of 40 type 0 persons whose lymphocytes were stimulated with red cell B antigen, only 20% produced plaques. Similarly, reciprocal stimulation of type A and B persons showed higher plaque formation (65%) when 23 type B persons were stimulated with A than when 48 type A persons were stimulated with B red cells (19%). An optimal dilution of sensitizing cells is necessary and maximum time of sensitization was approximately 7 days. Received for publication 17July, revised, accepted 9 October 1978

When studying the immune response of gen (PWM), peripheral blood lymphocytes humans, peripheral blood lymphocytes are from normal persons can be induced to the most readily available source of cells. produce specific plaque-forming cells Although it appears that peripheral blood (PFC). B lymphocytes produce heterophile antibodies to sheep red blood cells (Luzzati et Material and Methods al. 1976,1977, Mann & Falk 1973), plaqueforming antibodies to human red cells have Peripheral blood lymphocytes were isolated not yet been produced. Without deliberate over Ficoll-Hypaque from healthy adults. immunization of humans, the natural anti- Five million lymphocytes were cultured in body formation to anti-A and anti-B red 25 ml plastic flasks (Falcon #3031) in 5 ml cell antigens should be of value in studying of RPMI/1640 medium. To sensitize the allogeneic antibody formation. It will be lymphocytes, washed red blood cells of shown here that, following in vitro immu- defined ABO types were used. Only Rh nization in the presence of pokeweed mito- positive red cells were utilized. PWM

' This work was supported in part by grant AM 01375 from the National Institute of Arthritis and Metabolic Diseases. 0001-2815/79/010189-06 $02.50100 1979 Munksgaard, Copenhagen

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(Gibco) was used at different concentrations. PWM was dissolved in distilled water and kept at 4OoC and used within 30 days. The fetal calf serum (Reheis Chemical Co.) was added to the media at 20% concentration after heat inactivation at 56OC for 30 min. During sensitization, the cultured lymphocytes were incubated for 310 days at 37'C in a 5% COz incubator. At the end of the sensitization period, during which time the cells were left undisturbed, the cells were washed twice and adjusted to a count of 2 million cells per ml. The plaque-forming assay was performed essentially according to Cunningham & Szenberg (1967). 0.2ml of the lymphocyte suspension, 0.02 ml of 50% indicator red cells, and 0.2ml of absorbed rabbit complement were mixed. The rabbit complement was absorbed in the cold with pooled human A, B, and Rh D RBC. The mixtures were put into the chambers and incubated at 3 7 ' ~for 90-120min. AU experiments were done in duplicate. Hemolytic plaques were counted with lOOX magnification.

Results Lymphocytes from normal persons who were type A, B, or 0 were tested against red cell type A or B in the plaque-forming assay. In a total of 20 type A, 21 type B, and 24 type 0 lymphocytes tested, no plaques were found. Thus, despite the presence of natural antibodies, peripheral blood lymphocytes did not produce plaques when tested directly. Presensitization in the presence of PWM. In order to generate PFC, lymphocytes had to be incubated with the target red cells in the presence of PWM. It was possible to show that the PFC that were induced were antigen specific by the use of A and B target cells. As shown in Table 1, lymphocytes from a person with red cell type A were incubated with B red cells in the presence or absence of PWM. N o PFC were found with A red cell targets, but 33 PFC/106 cells were found against red cell type B targets. With PWM alone, no plaques were formed. With lymphocytes from a type B donor, sensitization in the presence of

Table 1 Generation of PFC b y in vitro Sensitization and pokeweed mitogen

Blood Type of Lymphocyte Donor

Sensitizing RBC Type

A

B B -

B

A A -

0

A A B B

PWM

PFC/106 Cells

HA Titers

A RBC

B RBC

+ + +

42 0 11

0 0 0

+

52

2 0 36 0 0

+

-

i-

i-

0 16 0 2

Anti-A 1:16

Anti-A 1:8 Anti-B 1:8

The numbers of PFC/million cells are given for different combinations of respondkg lymphocytes sensitizing cells - and pokeweed mitogen. The hemagglutinating titers of plasma of the lymphocyte donor are given.

191

HEMOLYTIC PLAQUE FORMATION

PWM resulted in 42 PFC/106 cells against A red cells but no PFC against B red cells. Of some interest was the induction of PFC against type A red cells to a smaller extent by PWM without the presence of A red cells. This could be attributed to stimulation of memory cells since PFC were not produced against B red cells. With lymphocytes from a blood type 0 person, PFC against A or B were induced by incubation with red cells. Slight nonspecific stimulation of PFC to red cell type A was seen after incubation with red cell type B, although this could be the restimulation effect of PWM. Time course of PFC generation. The effect of different incubation times necessary for sensitization was tested in the presence of PWM. As given in Table 2, PFC were first detectable at 5 days, reached a maximum generation after 7 days’ sensitization, and were negative after 9 days’ incubation. Most importantly, the PFC generation can

be seen to be a specific one, that is, they are only produced against the sensitizing red cell. Sensitizing red cell concentration. The number of sensitizing red cells appears to have an optimum. High concentrations tend to decrease the number of PFC as do lower concentrations of sensitizing cells (Table 3). With the first blood type 0 lymphocyte donor, stimulation with red cell type A in different concentrations produced PFC that reacted only slightly to B indicator cells but strongly to A indicator red cells. In the second blood type 0 lymphocyte donor, stimulation again with red cell type A produced some PFC against B indicator red cells. At certain sensitizing antigen concentrations, the PFC to A red cells clearly predominated: 6 9 PFC versus 21 and 141 versus 14. However, at other concentrations similar numbers of PFC to A and B targets were noted. The specificity of the response may thus be influenced by the concentration of sensitizing cells.

Table 2 Duration of in vitro sensitization and generation of PFC

Blood Type of LymphocyteDonor

PFC/106 Cells Incubation Time (Day) 5 6 7

Sensitizing RBCType

Indicator RBCType

1

3

A

A B

0 0

2

10

30

192

0

6

0

0

-

A B

0 0

0 0

0 5

0 0

34

2

0

0

0

0

A

0 0

0 0

20

105

5

120 0

18 0

0

0

12

0

A 0

B

8

9

30 0

0 0

0

A B

0 0

0 0

24

20

9

0

3

0

0

0

0

A

A B

0 0

0 0

10 0

18 4

107

70

0

0

0

0

-

A B

0

0

0

8 0

19 1

18 3

18 2

0

0

-

0

0

The number of PFC generated after different periods of presensitization in the combinations given is shown.

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SASAKI AND TERASAKI

Table 3 Concentration of sensitizing red cells and plaque -forming cells Blood Type ofLymphocyteDonor

Sensitizing RBCType

0

A

0

A

A

B A

B

Indicator RBCType

1%

PFC/1ob Cells Sensitizing RBC Concentration 0.1% 0.01% 0.001%

A

2

142

220

B

4

10

5 __

B

7 10

69 21

B A

0 0

0 10

~-

A

~

0

112 2

17 3

141 14

28 13

14 11

40 102

0

0

5

0

-~

PWM dose effect on PFC generation. PWM plaques, that is more than 50 plaques per in dilutions of 1:1 to 1:20 did not produce million cells, occurred mainly with type A a marked difference in PFC generation. targets (19% of type 0 lymphocytes stimuHowever, in dilutions above 1:20 there was lated with A and 26% of B lymphocytes a marked loss in PFC production. At 1: 80 stimulated with A). In contrast, only one essentially n o PFC could be generated type 0 lymphocyte stimulated with B (2.5%) produced more than 50 plaques and (Table 4). only one type A lymphocyte stimulated Summary of the PFC formed after in vitro with B (2%) producedmore than 50 plaques sensitization. The greater proportion of per million cells. PFC generated by the red cell A antigen as Discussion compared to the red cell B antigen is eviFrom the data presented, it appears that dent from Table 5. That is, the PFC PFC can be produced against the human A response was greater when using red cell B red cell antigens from peripheral and type A than red cell type B as targets. With type 0 lymphocytes, 52% produced plaques blood lymphocytes. This first demonto type A whereas only 20% produced stration (to our knowledge) of PFC to A plaques to type B red cells. Also, 65% of and B antigens should lay the groundwork lymphocytes from type B persons produced for many studies using this simple indicator plaques to type A whereas only 19% of antigen. As detailed here, PWM and in vitro type A lymphocytes produced plaques to sensitization have been found to be necesstype B. In addition, large numbers of ary. Despite the presence of circulating Table 4 PWM dose effect on generation of PFC Blood Type of Lymphocyte Donor

Sensitizing RBC Type

Indicator RBC Type

0

A

0 B

B

A B

A

A

B

A B

1:l

PFC/106 Cells PWM Dilutions 1:10 1:20 1:40

78 53 62 26

118 66 76 36

89 25 84 54

17 0 13 0

1:80

2 0 0

0

193

HEMOLYTIC PLAQUE FORMATION Table 5 Summary of PFC formation after in vitro sensitization Blood Type of Lymphocyte Donor

Sensitizing RBC Type No.

0 0 A B

A B

A

A

B

B

B

A

91 40 48 23 9 10

PFC/106 0

44(48%) 32(80%) 39(81%) 8(35%) 9(100%) 10(100%)

1-10

11-50

51-100 101-200

11(12%) 19(21%) 7(8%) 3(7.5%) 4(10%) 1(2.5%) 4(8.5%) 4(8.5%) 1(2%) 2(9%) 7(30%) 4(17%)

5(5.5%)

>201

5(5.5%)

2(9%)

Type A and B red cells were used for sensitization and as targets. The numbers of combinations with different ranges of PFC are given together with the percentage distribution. For example, 91 samples from type 0 donor lymphocytes were sensitized with type A red cells and 44 (or 48%)did not produce plaques and 11 (12%) produced 1-10 plaques per million cells.

anti-A and anti-B antibodies, the lymphocytes from peripheral blood did not produce plaques when tested directly, which indicates that the circulating lymphocytes are not those producing the antibody. However, after 5-7 days of in vitro sensitization of the lymphocytes, PFC were produced. This is probably a secondary response If memory cells. PWM by itself induces a low level of PFC, but these cells are almost invariably directed against indicator cells to which antibodies exist in the serum of the lymphocyte donor. To what extent nonresponse is attributable to technical factors in the in vitro sensitization or to inherent responsiveness has not yet been determined. Certainly one factor identified in this study is the antigen, for response to type A red cells was markedly superior to response to type B red cells. One explanation of the variability in response is in the purification of lymphocytes from blood. Different preparations from Ficoll-Hypaque separation could contain different proportions of “helper” T lymphocytes which are apparently needed in B-cell activation (Watanabe et al. 1974, Gmelig-Meyling et al. 1977, Keightley e t al.

1976). If monocytes are needed (Watanabe et al. 1974, McDougal & Gordon 1977, Calderon et al. 1975), the numbers present may also be varied in the different suspensions. The test system described here also appears to be potentially useful for tolerance studies, for it has been shown that excess antigenic stimuli result in lower numbers of PFC. Inhibition of PFC generation by increased doses of red cells of other specificities should be of interest. Other specificities, such as Rh, Kell, MNS, etc., present on the red cells may function as alloantigens. They may also act synergistically with the ABO antigens. The cumulative disparity between the stimulator and responder may be a factor in PFC generation. Immunologic responsiveness as determined by immune response (IR) genes may also play an important role in whether a given cell responds to defined antigens. Although a slight tendency of HLA-B44 persons to be nonresponders was noted, larger scale studies together with reproduci bility tests will be required to establish linkage of this in vitro responsiveness measure with HLA specificities.

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A cknowledgernent We thank Miss Beverly Looman for her

excellent technical assistance.

References Calderon, J., Kiely, J. M., Lefko, J. L. & Enanue, E. R., (1975)The modulation of lymphocyte functions by molecules secreted by macrophages. I. Description and partial biochemical analysis.J. exp. Med. 142,151. Cunningham, A. J. & Szenberg, A. (1967)Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599. Gmelig-Meyling, F., Uytdehaag, A. G. C. M. & Ballieux, R. E., (1977)T cell-dependent pokeweed mitogen-induced differentiation of blood B lymphocytes. Cell. Immunol. 33, 156. Keightley, R. G., Cooper, M. D. & Lawton, A. R. (1976)The T cell dependence of B cell differentiation induced by pokeweed mitogen. J. Immunol. 117,1538. Luzzati, A. L., Hengartner, H. & Schreier, M. H.

(1977) Induction of plaque-forming cells in cultured human lymphocytes by combined action and EB virus. Nature 269,419. Luzzati, A. L., Taussig, M. J., Meo, T. & Pernis, B. (1976)Induction of an antibody response in cultures of human peripheral blood lymphocytes. J. exp. Med. 144,573. Mann, P. I. & Falk, R. E. (1973) Production of antibody by human peripheral lymphocytes after allogeneic stimulation. In Seventh Leucoc y t e Culture Conference, ed. Daguillard, F., p, 39. Academic Press, New York, London. McDougal, J. S. & Gordon, D. S. (1977)Generation of T helper cells in vitro. I. Cellular and antigen requirements. J. exp. Med. 145, 676. Watanabe, T., Kazuyuki, Y., Yagura, T. & Yamarnura, Y., (1974)In vitro antibody formation by human tonsil lymphocytes. J. Immunol. 113. 608. Address Paul I. Terasaki, Ph.D. 1000 Veteran Avenue Los Angeles, CA 90024 U.S.A.