Tissue Antigens (1976), 8, 151-155 Published by Munksgaard, Copenhagen, Denmark N o part may be reproduced by any process without written permission from the author(s)

SHORT COMMUNICATION

Typing for HLA-D: Primed LD Typing and Homozygous Typing Cells

Fritz H. Bach’, Elizabeth A. Valentine’, Barbara J. Alter’, Arne Svejgaard‘ and Mogens Thomsen’

‘Immunobiology Research Center and Departments of Medical Genetics and Surgery,

University of Wisconsin, Madison, Wisconsin, U.S.A.; and ‘Tissue Typing Laboratory of the Blood Grouping Department,

State University Hospital, Copenhagen, Denmark

Received for publication 29 December 1975, accepted 13 May 1976

Until recently, the only method of evaluat- ing the relationship for HLA-D between two individuals has been the primary mixed leukocyte culture (MLC) test. The descrip- tion by Bradley et al. (1972) of the use of homozygous typing cells (HTC’s) for the definition of major histocompatibility com- plex (MHC) LD determinants in pig has now been considerably extended by studies in man (Mempel et al. 1973, van den Tweel et al. 1973, Jergensen et al. 1973, Dupont et al. 1973a).

Lymphocytes “sensitized” in MLC and subsequently restimulated, undergo a rapid, secondary-type proliferative response (Anderson & Hayry 1973, Zier & Bach 1975, Fradelizi & Dausset 1975, Sheehy et al. 1975a). We have proposed (Sheehy et al.

1975a,b, Bach et al. 1975) that restimulation of this proliferative response can be used for HLA-D typing in unrelated individuals (primed LD typing or PLT) when one- haplotype-different individuals are used in the initial sensitization for this preparation of the PLT cells. Fradelizi & Dausset (1975) suggested in their paper that restimulation is specific for the sensitizing cell used in the primary MLC, and made no suggestion that this accelerated proliferative response could be used for HLA-D typing. The present study is an attempt to correlate results obtained using HTC’s and PLT cells. The data suggest that these methods both mea- sure HLA-D although certain differences are of interest.

This work was supported by NIH grants AI-11576, CA-16836, CA-14520 and National Foundation- March of Dimes grant CRBS 246 and grants from the Danish Medical Research Council, the Daell Foundation, the Danish Cancer Society, and the Foundation for Medical Research in Copenhagen, Greenland and the Faroe Islands. This i s paper no. 1943 from the Laboratory of Genetics and paper no. 70 from the Immunobiology Research Center, the University of Wisconsin, Madison, Wisconsin.

152 BACH ET AL.

table 1 Restimulation of HLA-DW2 specific PLT cells by p e r s m phenotypically “homozygous”, “heterozygous” or

“negative” for DW2

Stimulating PLT responding cells cells A/RR, NKJX NBA, CIRR, CJKJX C/BA,

A C BB -1-

BBJ

RR DW2JDW2 KJ

BA

M T Cl

DW2/-

AR

Medium control

588 4,719 3,097 2,472

20,778 21,110 22,744

13,059 12,997 15,195 12,745

123

734 3,965 3,752 2,390

21,550 22,903 22,749

1435 1 14,402 16,983 13,385

414

886 6,523 4,347 1,745

24,957 24,385 25,188

16,524 16,845 18,737 14,392

353

11,517 1,695 5,969

1 1,607

40,110 41,974 43,827

30,656 31,471 31,77 1 30,005

165

11,223 1,703 5,013

10,362

44,821 48,818 45,208

30,842 34,322 3 1,785 33,009

433

6,036 1,699 3,559 6,479

29,802 30,094 32,281

20,377 24,041 23,189 22,475

404

Numbers are mean cpm tritiated thymidine uptake from triplicate cultures of 5 x lo4 PLT responding cells and 2X lo5 stimulating cells. Average percent standard deviation is 12.1.

Materials and Methods Cells A and C (see Table 1 ) were from individuals in Madison. All other cells were shipped as whole blood at ambient tempera- ture from Copenhagen where they had been previously typed for HLA-D with HTC’s (Thomsen et al. 1975). Lymphocytes were obtained from peripheral blood by Ficoll- Hypaque density gradient centrifugation and were used either fresh or after being frozen in liquid nitrogen (Segall & Bach 1973).

Primary MLC tests (Hartzman et al. 1971) were performed with fresh cells. PLT test (Sheehy et al. 1975a) results for Table 1 used fresh cells of A and C as responding cells primed with HTC’s that had been frozen in liquid nitrogen. All restimulating cells had been likewise frozen. For data presented in Table 2, the experiment was performed in Copenhagen and all cells were fresh.

Results We tested four DW2 HTC‘s (HB, RR, KJ,

and BA) in primary MLC with 12 different responding cells including cells of the four HTC donors. Four responders, A, C, BB and BBF incorporated between 24,854 and 79,713 cpm (results not shown) and are thus DW2 negative. Cells of individuals BKJ, M T , CJ and AR are proven heterozygotes for the DW2 cluster; their cells incorporated between 1,429 and 1 1,185 cpm with a mean of 3,721 cpm. The four HTC‘s tested against one another incorporated between 241 and 1,073 cpm. Cells of individual A were sen- sitized in three separate flasks to each of three HTC‘s: RR, KJ and BA; cells of individual C were similarly sensitized. The six different PLT cells were tested for their response to the restimulating cells of the same individuals used above as responding cells. The results are presented in Table 1 . Two findings are of note: First, using any given PLT cell, there is clear discrimination (no overlap in these mixtures) between the results obtained using DW2 negative and DW2 positive individuals as donors of

Tabl

e 2

Res

timul

atio

n of

HL

A-D

W4

and

HL

A-D

WI

spec

ific P

LT

cells

by c

ells

from

fam

ily m

embe

rs a

nd th

ree

unre

late

d pe

rson

s

Res

timul

atin

g ce

lls

2 $ Fa

ther

M

othe

r C

hild

1

Chi

ld 2

P

M

L M

ediu

m

DW

1/10

7? D

WI/

DW

4? D

W l/

DW

l 10

7?/D

W4

DW

I/D

WJ

DW

2/D

W5

DW

4/10

7 co

ntro

l

Hap

loty

pe

reco

gniz

ed

Com

bina

tion

0

PLT

1 (F

athe

r) (M

othe

r),

DW

4 0

4793

16

50

4887

99

3 54

5 1

5744

13

2 2

(Mot

her)

(C

hild

l),

D

W 1

1298

0

1615

10

0 84

32

10

0 27

0

P, M a

nd L

are

unr

elat

ed t

o th

e fa

mily

. R

esul

ts e

xpre

ssed

as

med

ian

cpm

14C

-lab

eled

thym

idin

e up

take

fro

m t

ripl

icat

e cu

lture

s of

5x

lo4

resp

ondi

ng c

ells

and

2X

lo5

stim

ulat

ing

cells

. Q

uest

ion

mar

ks i

ndic

ate

bord

erlin

e H

LA-D

ty

ping

res

pons

es w

ith H

TC

’s.

Bac

kgro

und

cpm

(re

stim

ulat

ion

with

ori

gina

l re

spon

ding

cel

l) su

btra

cted

fro

m e

ach

rest

imul

atio

n va

lue.

154 BACH ET AL.

restimulating cells. Second, there is a gene dosage effect in that, with any given PLT cell, restimulating cells of individuals homozygous for DW2 stimulate more than cells of individuals heterozygous for this cluster.

Table 2 shows the results of testing with two PLT cells established within a family that had been typed for HLA-D with HTC’s. The first defines the DW4 cluster within that family assuming that the DW 1 haplotype of father and mother are identi- cal. The cells are stimulated with cells of each member of the family plus cells of three unrelated individuals, one of whom is posi- tive for DW4 by HTC testing. Results of PLT show that in each case where a cell carries the DW4 cluster, restimulation was of the same order as that obtained with the specific initial sensitizing cell. However, in addition, cells of individual M, which by HTC testing did not carry the DW4 cluster, restimulated to approximately the same extent as did cells carrying that cluster. For the second PLT cell, cells of the mother are sensitized to the HTC of child 1 . (This behaves as an HTC in this family and in the unrelated population; this was confirmed in the International Workshop when measur- ing DWl (Thomsen et al. 1975).) Cells of child 1 and the father restimulate signific- antly.

Discussion The results presented in this paper confirm and extend the findings of Hirschberg et al. (1976) showing a correlation between HTC’s and PLT cells. Our findings demonstrate that PLT cells sensitized against HLA-D determinants on an HTC can recognize those determinants on an unrelated restimulating cell.

Evidence has been obtained that a single HLA haplotype can code for several HLA-D determinants (Dupont et al. 1973b, Bach et al. 1975). Individuals deemed heterozygous

for an HLA-D cluster as determined by HTC’s, may in fact not carry all of the HLA-D determinants present on that HTC (Thomsen et al. 1976). The findings shown in Table 2 are consistent with this concept. The fact that cells of individual M restimu- late PLT-DW4 to approximately the same degree as the specific sensitizing cell could be explained on the basis that M cells carry, in heterozygous or homozygous form, enough of the HLA-D determinants which make up the DW4 cluster to cause extensive restimulation. Models could be proposed to explain why M’s cells are nonetheless nega- tive with DW4 HTC‘s. Such model building, at the moment, seems unwarranted since further testing with these two methods will no doubt resolve this question. Results with PLT-“DW1” suggest that the father’s DW 1 haplotype has at least one extra LD deter- minant as compared with DW1 haplotype of the mother, even though both cells gave typing responses with DW 1 HTC’s. To the extent that the interpretation of multiple LD determinants associated with a single hap- lotype is correct and restimulation is caused by HLA-D determinants (Sheehy et al. 1975a,b, Mawas et al. 1975, Bradley et al. 1975), the use of the word “homozygous” for phenotypically HTCs, while useful, is, at least in some cases, something of a ter- minological inexactitude.

References Anderson, L.C. & Hayry, P. (1973) Specific

priming of mouse thymus-dependent lympho- cytes to allogeneic cells in vitro. Eur. J . Immunol. 3, 595-599.

Bach, F.H., Sondel, P.M., Sheehy, M.J., Wank,R., Alter, B.J. & Bach, M.L. (1975) The complexity of the HL-A LD system: A PLT analysis. Histocompatibility Testing 1975, p. 576-580. Munksgaard, Copenhagen.

Bradley, B.A., Edwards, J.M., Dunn, D.C. & Calne, R.Y. (1972) Quantitation of mixed lym- phocyte reaction by gene dosage phenomenon. Nature New Biol. 240, 54-56.

TYPING FOR n u - D 155

Bradley, B.A., Sheehy, M., Keuning, J.J., Ter- mijtelen, A., Franks, D. & van Rood, J.J. (1975) The phenotyping of HLA-D region products by negative and positive mixed lymphocyte reactions. Immunogenetics (submitted for publi- cation).

Dupont, B., Jersild, C., Hansen, G.S., Staub Nielsen, L., Thomsen, M. & Svejgaard, A. (1973a) Typing for MLC determinants by means of LD-homozygous and LD- heterozygous test cells. Transplant. Proc. 5 , 1543-1 550.

Dupont, B., Jersild, C., Hansen, G.S., Staub Nielsen, L., Thomsen, M. & Svejgaard, A. (1973b) Multiple MLC (LD) determinants on the same HL-A haplotype. Transplant. Proc. 5, 1481-1487.

Fradelizi, D. & Dausset, J. (1975) Mixed lympho- cyte reactivity of human lymphocytes primed in vitro. I . Secondary response to allogenic lym- phocytes. Eur. J. Immunol. 5 , 295-301.

Hartzman, R.J., Segall, M., Bach, M.L. & Bach, F.H. (197 1) Histocompatibility matching VI. Miniaturization of the mixed leukocyte culture test: a preliminary report. Transplantation 2 ,

Hirschberg, H., Kaakinen, A. & Thorsby, E. (1976) Typing for HLA-D determinants. Com- parison of typing results using homozygous stimulating cells and primed lymphocyte typing PLT. Tissue Antigens 7 , 213-220.

Jcirgensen, F., Lamm, L.U. & Kissmeyer-Nielsen, F. (1973) Mixed lymphocyte cultures with inbred individuals: an approach to MLC typing. Tissue Antigens 3, 323-339.

Mawas, C., Charmot, D.& Sasportes, M. (1975) Secondary response of in vitro primed human lymphocytes to allogeneic cells. I. Role of HL-A antigens and mixed lymphocyte reaction stimulating product in secondary in uitro pro- liferative response. Immunogenetics 2 , 449463.

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

268-273.

determinants ising homozygous and heterozygous rr erence cells. Transplant. Proc.

Segall, M. & Bach, M.L. (1973) Use of C V O -

preserved leucocytes in mixed leucocyte cul- ture. Cryopreseruation of Normal and Neoplastic Cells, ed. Weiner, R.S., Oldham, R.K. & Schwartzenberg, L., p. 107-1 14. INSERM, Paris.

Sheehy, M.J., Sondel, P.M., Wank, R., Bach, M.L. & Bach, F.H. (1975a) H L A LD typing: a rapid assay using primed lymphocytes. Science 188,

Sheehy, M.J., Sondel, P.M., Bach, F.H., Sopori, M.L. & Bach, M.L. (1975b) Rapid detection of LD determinants: the PLT assay. Histocom- patibility Testing 1975, p. 569-575. Munks- gaard, Copenhagen.

Thomsen, M., Jacobsen, B., Platz, P., Ryder, L.P., Staub Nielsen, L. & Svejgaard, A. (1975) LD- typing, polymorphism of MLC determinants. Histocompatibility Testing 1975, p. 509-518. Munksgaard, Copenhagen.

Thomsen, M., Morling, N., Platz, P., Ryder, L.P., Staub Nielsen, L. & Svejgaard, A,. (1976) Specific lack of responsiveness to certain HLA-D (MLC) determinants with notes on primed lymphocyte typing (PLT). Transplant. Proc. (in press).

van den Tweel, J.G., Blusse van Oud Alblas, A., Keuning, J.J., Goulmy, E., Termijtelen, A., Bach, M.L. & van Rood, J.J. (1973) Typing for MLC (LD). I. Lymphocytes from cousin- marriage offspring as typing cells. Transplant. Proc. 5, 1535-1538.

Zier, K.S. & Bach, F.H. (1975) Secondary responses of human lymphocytes to alloanti- gens in vitro. Scand. J . Immunol. 4, 607-61 1.

5, 1529-1534.

1308-1 3 10.

Address: Fritz H. Bach Immunobiology Research Center Univetsity of Wisconsin Madison, Wisconsin 53706 U.S.A.