Tissue Antigens (1975), 7, 213-219 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Typing for HLA-D Determinants. Comparison of Typing Results using Homozygous Stimulating Cells and Primed Cultures

Henry Hirschberg, Aulikki Kaakinen and Erik Thorsby Tissue Typing Laboratory, National Hospital of Norway, University Hospital, Oslo, Norway

Typing for HLA-D determinants, using primed lymphocyte typing (PLT), has been compared to the results obtained using conventional homozygous stimulating cell primary MLC tests. T h e HLA-Dwl, -Dw2 and -Dw3 specificities were studied. Preliminary results indicate that these two methods essentially type for the HLA-D determinants and that reproducible results can be obtained employing the PLT technique after 24 hours of incubation in the secondary cultures. Received for publication 20 October, revised, accepted I 9 December 1975

A positive response in mixed lymphocyte cultures (MLC) is governed by genetic differences at the HLA-D locus‘ (Yunis & Amos 1971, Eijsvoogel et al. 1972). Reports from several laboratories have demonstrated the possibility of typing for HLA-D determinants by means of the primary MLC test employing homozygous stimulating cells (Mempel et al. 1973, van den Tweel et al. 1973, Jorgensen et al. 1973). In this way a number of HLA-D determinants have already been identified (Thorsby & Piazza

Recently, an alternative method of typing for HLA-D determinants was suggested, employing “ p r i m e d o r secondary lymphocyte cultures (Sheehy et al. 1975, Fradelizi et al. 1975): the primed lymphocyte typing (PLT) test. These authors demonstrated that between family members the secondary response was dependent upon the HLA-D disparity in the primary MLC combination. On the other hand, typing for HLA-D determinants using unrelated donors proved more problematic (Fradelizi et al.

1975).

1975).

’ Previously called LD-1 or the MLC-S locus.

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HIRSCHBERG ET AL.

T h e basis of the PLT test in man (originally demonstrated in the mouse, Hayry & Anderson 1974), is that responding lymphocytes are first “primed” by stimulating them with mitomycin-treated cells from a n individual differing for HLA-D determinants. During this initial incubation of 10-14 days the primary MLC response is completed. When the “primed” responding cells are restimulated in secondary mixed lymphocyte cultures by the original stimulating cells, a proliferative response with accelerated kinetics and increased magnitude develops (Fradelizi & Dausset 1975). Ideally restimulation with lymphocytes not possessing the HLA-D determinant for which the cells are primed, gives a relatively feeble response, while cells sharing the HLA-D determinant with the original stimulating cell donor gives a response of the same order of magnitude as that elicited by restimulation with the original stimulator, provided the cultures are harvested relatively early. T h e advantages of the PLT test are primarily that the response is elicited after 1-2 days compared to 5 days for homozygous typing and that rare HLA-D determinants for which no homozygous typing cells presently exist can be typed for by “priming” combinations within families (Sheehy et al. 1975). In the experiments reported here we have compared the two methods for the identification of the HLA-Dwl, -Dw2 and -Dw3 determinants. In unrelated individuals the results indicate that under proper conditions and in chosen combinations, the primed lymphocyte typing technique gives results in accordance with those obtained in primary MLC tests using homozygous stimulating cells. Materials and Methods O u r method for MLC typing with homozygous cells has been reported in detail elsewhere (Thorsby et al. 1975).

Previously typed individuals were selected on the basis of their HLA-D determinants and primary cultures were established in 10 ml tissue culture tubes (Falcon Plastics) employing 2 x lo6 responding lymphocytes in combination with 4 x lo6 mitomycin treated lymphocytes in 4 ml of RMPI 1640 medium, supplemented with antibiotics and 20% normal human serum. T h e cultures were incubated at 37°C in a 95% air and 5% C 0 2 atmosphere for 9 to 10 days, the medium changed once on day 6. After this “priming” incubation, the cells were washed once, counted and redistributed as triplicate cultures into the wells of microtiter plates (Cookes) at a concentration of 5 x lo4 in 0.1 ml culture medium. Stimulating lymphocytes from a panel of previously HLA-D typed donors were freshly separated and 5 x lo4 cells were added to each well resulting in a final total volume of 0.2 ml. T h e stimulating cells were not mitomycin-treated since the secondary restimulation cultures were assayed after 24 and 48 h where no significant proliferation of the non-primed stimulating cells occurred. Control experiments comparing the results in secondary restimulation cultures for both mitomycintreated and untreated stimulating cells showed no significant differences. Parallel cultures were employed and were harvested after 24 and 48 h of incubation, following a 12 h pulse of 3H-thymidine. Results T h e results of secondary restimulation of cultures primed for the HLA-Dw2 determinant are presented in Tables 1 and 2. In the first experiment (Table l ) , the initial stimulating cell donor was typed (by homozygous typing cells) to possess the Dw2 determinant and an unknown HLA-D determinant. This individual was not HLA-B7 positive, an antigen known to have a high non-random association with the Dw2 determinant. T h e other stimulating cell

215

T Y P I N G FOR HLA-D DETERMINANTS

Table 1 Response of cultures primedfor the HLA-Dw2 determinant. Heteroiygous stimulation in the primary culture‘”

Stimulating cell donor

X A B C D E F G H I

-

Table 2 Response of cultures Primed for the D w 2 determinant. Homozygow stimulation i n the primary culture‘n’

Stimulating Secondary restimulation cell donor(b) Response median cpm 24 h(C)

Secondary restimulation response (median cpm(b) SE)

Dw2,Dw2,Dw4 Dw2,Dw4 Dw2,Dw3 Dw2,Dw3 Dw2,Dw3 Dw2,Dw3 Dw6,Dwl, Dw2,Dwl

24 h

48 h

10370 -I 960 12531 2 875 11682 2 1232 1 4 0 8 5 5 816 8 9 3 9 2 827 1 5 6 1 9 5 720 13737k 630 4 1 7 7 k 215 2 1 2 2 t 312 16797&1018

15791 2 1670 16235% 875 16865% 815 1 9 8 1 4 2 615 15816+ 1497 2 4 2 9 9 t 1715 17430% 809 1 1 1 0 2 t 222 8 0 1 2 t 570 20648 _t 1063

Y A B C D E F G H I

- Dw2,Dw2

(a)

Primary MLC combination LYm: L: HLA-AZ, 10; B7; Dwl Y: HLA-A2, B7, Dw2lA2, B7, Dw2. The same donors were used as shown in Table 1. Autologous control values of 492 have been subtracted.

~~~

(a)

(b)

Primary MLC combination LXm: L: HLA-A2, 10; B7; Dwl X: HLA-A2, 11; B5, 12; Dw2 The autologous control counts of 336 and 238 cpm at 24 and 48 h, respectively have been subtracted from these values.

(b) (‘)

-

18875_+1420 12672k 920 1 1 0 2 1 k 430 14152k 864 10370k 517 13052+1311 1 0 5 7 3 k 615 3362+ 219 1927+ 74 1 2 3 7 5 5 415

Dw2,Dw4 Dw2,Dw4 Dw2,Dw3 Dw2,Dw3 Dw2,Dw3 Dw2,Dw3 Dw6,Dw1,Dw2,Dwl

Table 3 Response of cultures primed for the HLA-Dw3 determinant. Heterozygous stimulation in the primary culture

Secondary restimulation response (median cpm SE) Primary comb. LXmta) Primary comb. LYm 24 h 48 h 24 h 48 h

Stimulating cell donor

X Y A B C D E F

-

Dwl,Dw3 Dwl,Dw3 Dw2,Dw4 Dwl,Dw3 Dw2,Dw3 Dw2,Dw3 Dw2,Dw4 Dwl.Dw2

382 387 295 283 317 266 472 307

27 ? 42 C 22 2 15 f 18 t 32 f_ 19 t 31 _+

~ _ _ _ _ _ _

(a)

L - HLA-A2, 10; 1O;lO; B7; Dwl. X - HLA-A1, 2; B5, 7; Dwl, ~ 3 . Y

- HLA-A1;

B5, 8; Dwl, w3.

6215 5975 3025 5398 6424 6253 3310 2161

k 325 5 415 ? 281 -C 212 5 514 2 319 2 372 k 315

402 403 363 350 405 343 439 309

2 38 ? 22 ? 15 k 17

t 32

2 37 +_

21

k 18

6526 6892 4455 5232 6813 6672 4299 2245

k

473 608 2 294 C 272 C 437 t 609 k 215 k 176

k

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HIRSCHBERG ET AL.

donors were all previously typed more than once and many were HLA-D identical with each other as determined by homozygous typing and reciprocal MLC tests. As can be seen from Table 1, after 24 h a full proliferative response is elicited. T h e response to many of the stimulating cells possessing the Dw2 determinant was, in fact, greater than that observed towards the original stimulating cell donor and was never less than (approximately) 85% of this value. On the other hand, the two donors not possessing the Dw2 determinant gave significantly less stimulation, although greater than autologous controls. T h e picture is slightly less clear at 48 h of culture. T h e HLA-A and -B typing data are not shown, but no clear correlation could be seen between these antigens and the results obtained. In the experiment shown in Table 2, a cell donor homozygous for the Dw2 determinant was used as the initial stimulating cell donor. T h e cells were obtained from the Dw2 hornozygous donor originally used to type the panel. The secondary cultures were harvested after 24 h. As seen in the Table, restimulation with the initial stimulating cell donor gave, in this case, a greater response than any of the heterozygous panel donors. T h e discrimination between the HLA-Dw2 positive and negative donors was in this experiment greater than in the previous one, with a clear bimodal distribution of the responses. T h e HLA-Dw3 determinant was also studied in three separate primed cultures. Two different heterozygous stimulating cell donors were used with the same responding cell donor in two of the experiments. These donors, typed to be HLA-D identical (both possessed the Dw3 determinant), shared the Dwl determinant with the responding cell donor. One of the donors was also HLA-B8 positive, an antigen known to have a high non-random association with the Dw3 determinant. T h e results are shown in Table 3.

Both primed cultures had similar secondary restirnulation responses when challenged with a panel of previously typed donors, three of whom possessed the Dw3 determinant. After 24 h of incubation, no significant differences in the responses towards the Dw3 positive and negative stimulating cells could be observed, the level of response being only two to four times autologous control values. After 48 h, the

70 R e s p o n s e 120 110 0

100 90

ao 70 60

X

.

0

t

..

50 40 30

t

za

0

1c

0

0 DW3

DW1

Figure 1. Restimulation response at 24 h for primary cultures primed with Dw3 (x) or Dwl (O) homozygous stimulating cells. The same responding cell donor was used in both experiments. The secondary stimulating cells were HLA-D typed and four were Dw3 positive ( H ) and the other four Dwl positive ( 0 ). The results are shown as % of the response with restimulation by the homozygote cell used in the primary culture. Responding cell donor - HLA-A29, -B5, 14, D -I-.

Dw3 donor - HLA-Al, B8, Dw3/Al,B8,Dw3. Dwl donor - HLA-AS, B35, Dwl/A3,B35,Dwl.

217

TYPING FOR HLA-D DETERMINANTS

response to the Dw3 positive stimulating cell was clearly greater than that to the Dw3 negative cells although the latter group also gave significant stimulation. Thus, the discrimination between Dw3 positive and negative stimulating cells was of doubtful significance. N o convincing effect of the HLA-B8 antigen could be observed. Cultures were also primed with a cell homozygous for the Dw3 determinant. T h e results shown in Fig. 1 are for a panel of nine unrelated donors. After 24 h of secondary restimulation, a significant response was observed and in this case the homozygous original stimulating cell donor also gave the greatest response. The discrimination between Dw3 positive and negative donors was also reasonably good. After 48 h of incubation, the discriminative power was much poorer. Restimulation with a Dwl homozygous cell gave a response less than 5% of maximum value. Cultures were also primed for the Dwl determinant using a Dw 1 homozygous stimulating cell in the primary MLC. T h e results for one combination are shown in Fig. 1 . In this case there is also good agreement between the previous typing data using homozygous typing cells and the PLT test. In this experiment two of the heterozygous stimulating cells gave a greater response than restimulation by the homozygous cell donor used in the primary MLC. This may have been due to the fact that frozen homozygous cells were used in this experiment while the heterozygous stimulating cells were freshly separated. I n the experiments shown in Fig. 1, an identical panel of restimulating cells and the same responding cell donor was used in both experiments. T h e HLA-D specificity of the test is clearly demonstrated. These two primary cultures (primed for the Dw3 and Dw I determinants) were also restimulated for a second cycle using fresh Dwl and Dw3 homozygous stimulating cells. T h e cells were

separated from two new donors, i.e. not the same individuals used in the primary stimulation. T h e results for the tertiary cultures are shown in Fig. 2 using the same nine individuals who were employed in the secondary cultures (Fig. I). I n comparison between Fig. 1 and Fig. 2 a n increased discrimination between positive and negative typing results is clearly demonstrated in the tertiary cultures.

i', Response

I 0

80 8 8

60 50

-

40

-

30 -

10

20

:

m

0 0 0

mu

8

X

0

Dw3

Dwl

Figure 2. Tertiary restimulation response for primary and secondary cultures primed with Dw3(x) or Dw l(o) homozygous stimulating cells. Responding cell donor and restimulation panel as in Fig. 1. Dw3 and Dwl homozygous donors are two different individuals from those used in the primary stimulation.

218

HIRSCHBERG ET AL.

Discussion O n the basis of these preliminary experiments, it appears that primed cultures are capable of giving a specific early increased response to the HLA-D determinants studied. Full accord between typing results using homozygous cells in primary MLC typing and PLT tests primed with some of the same homozygous cells (Table 2, Fig. 1) was observed for the HLA-Dw 1, -Dw2 and -Dw3 determinants. However, the results for the Dw3 determinant, using heterozygous stimulating cells in the primary cultures (Table 3), are marginally significant. O n the other hand, the results shown in Table 1 for the Dw2 determinant clearly indicate that good discrimination between positive and negative cell donors can be obtained in cultures primed with heterozygous cells. This discrepancy may at least in part be due to the fact that the Dw3 determinant is at present less well defined than some of the other determinants (Thorsby & Piazza 1975, unpublished observations). T h e discriminative ability of the PLT test appeared to be dependent on the specific responding/stimulating cell donors used, certain combinations giving significantly better discrimination than others (unpublished observations). Restimulating the primary cultures for a second cycle seemed to increase the discriminative ability of the test, at least in some of the combinations (Fig. 2). The use of different homozygous cells possessing the same HLA-D specificity in the primary and secondary stimulation could conceivably reduce the background counts since non-HLA-D determinants are probably different on the different cells. Therefore, only the HLA-D responsive cells may be maintained in the cultures. Fradelizi et al. (1975) reported that the responding cell and the cells to be typed had to share a common HLA-D determinant in order to obtain reproducible results. This was not found to be strictly true in our

studies. T h e large background response seen against some of the stimulating cell donors, not sharing the HLA-D determi; nant with the priming cell may be due to the fact that the responding cells respond in a n accelerated fashion to all allogeneic stimulation. Unprimed cultures incubated for more than 9 days also seem to respond in an accelerated fashion (Fradelizi et al. 1975), therefore a portion of the accelerated response seems to be due to culture effects (cell synchronization) and does not specifically represent priming for HLA-D determinants. T h e role of other minor LD loci products could also be of importance for this background stimulation. T h e results given in Table 2 and Figs. 1 and 2 demonstrate a greater response in four of the five experiments when restimulation is performed with the priming HLA-D homozygous donor than with the heterozygous stimulating cell sharing the same determinant. This might indicate that a gene dose effect is detectable with the PLT technique. On the other hand, stimulation with homozygous cells not sharing HLA-D determinants with the initial stimulating cell gave a relatively feeble response in all of the combinations tested. We have found that the best typing discrimination is usually found after 24 h of incubation, w e have not, however, examined the period after 48 h since we feel that if the PLT test is to have clinical applications for necro-kidney donors, 24 h is an absolute limit using presently employed organ preservation techniques. Generally in our experiments, the discriminative ability of the ’ primed cultures degenerated with increased secondary restimulation incubation periods. T h e PLT test is at present only poorly defined. Further improvements are clearly required with the aim of increasing the discriminative ability of the test and decreasing the time required to obtain reproducible results.

TYPING FOR HLA-D DETERMINANTS

Acknowledgements

The authors would like to thank Anne Brit Thoresen and Astri Helgesen for excellent technical assistance and E. Garmann and K. Coyer for typing the manuscript. This work was supported by grants from the Norwegian Council for Science and the Humanities and Mr. Anders Jahre’s Medical Research Fund. References Eijsvoogel, V. P., van Rood, J. J., Toit, E. D. & Schellekens, P. T h . A. (1972) Position of a locus determining mixed lymphocyte reaction distinct from the known HL-A loci. Eur. J . fmmunol. 2, 413-418. Fradelizi, D. & Dausset, J. (1975) Mixed lymphocyte reactivity of human lymphocytes primed in vitro. I. Secondary response to allogeneic lymphocytes. Eur. I. Immunol. 5, 295-301. Fradelizi, D., Charmot, D., Mawas, C. F. & Sasportes, M . (1975) Secondary response of in vitro primed human lymphocytes to allogeneic cells. 111. Specificity for the mixed lymphocyte reaction stimulating determinants of the secondary proliferative response. Immunogenefics (in press). Hayry, P. & A n d e r s o n , L. (1974) Generation of T memory cells in one way mixed leukocyte culture. 11. Anamnestic response of secondary lymphocytes. Scand. J. Immunol. 3, 823-830. J@rgensen,F., Lamm, L.U. & Kissmeyer-Nielsen, F. (1973) Mixed lymphocyte cultures with inbred individuals: An approach to MLC typing. Tissue Antigens 3, 323-339.

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Mempel, W., Grosse-Wilde, H., Baumann, P., Netzel, B., Steinbauer-Rosenthal, I . , Scholz, S., Bertrams, J. & Albert, E. D. (1973) Population genetics of the MLC response: Typing for MLC determinants using homozygous and heterozygous reference cells. Transplant. Froc. 5, 1529-1534. Sheehy, M., Sonde], P. M., Bach, M. L., Wank, R. & Bach, F. H. (1975) HL-A LD typing: A rapid assay using primed lymphocytes. Science 188, 1308-1310. Thorsby, E. & Piazza, A. (1975) Joint Report from the Sixth International Histocompatibility Workshop Conference. 11. Typing for HLA-D (LD-I or MLC-S) determinants. Histocompatibility Testing 1975. Munksgaard, Copenhagen (in press). Thorsby, E., Helgesen, A., Rankin, B., M@ller,E. & Kaakinen, A. (1975) Identification of five lymphocyte activating determinants in man. Tissue Antigens 6, 147-160. van den Tweel, J. G., Blusse, van Oud, Alblas, A., Keuning, J. J.. Coulmy, E., Termijtelen, A., Bach, M. L. & van Rood, J. J. (1973) Typing for MLC (LD). 1. Lymphocytes from cousinmarriage offspring as typing cells. Transplant. Proc. 5, 1535-1538. Yunis, E. J. & Amos, D. B. (1971) Three closely linked genetic systems relevant to transplantation. Proc. nat. Acad. Sci. (Wash.) 68, 3031-3035. Address: H . Hirschberg Tissue Typing Laboratory National Hospital of Norway University Hospital Oslo 1, Norway