Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC)

Immunogenetics 6:91-100, 1978 [mmunogenetics �9 1978by Springer-Verlag New York Inc.

Serologieai Typing of HLA-D: Predietive Value in Mixed Lymphoeyte Cultures (MLC)

Dagfinn Albrechtsen, Anne Bratlie, Helena Nousiainen, Bjarte G. Solheim, Nina Winther, and Erik Thorsby

The Tissue Typing Laboratory, National Hospital of Norway (University Hospital), Oslo I, Norway

Abstract. Locally produced antisera and antisera received through the Seventh International Histocompatibility Workshop exchange were investigated for specific B-cell cytotoxic activity in a panel of 95 unrelated HLA-D-typed donors. A number of sera formed clusters defining eight B-cell specificities which were strongly associated (p < 0.001) to the HLA-D determinants Dwl-8. In panel investigations, only four triplets occurred. In rive HLA recombinant families, these B-cell specificities followed the HLA-B-D chromosomal region, and in one --B/D recombination, DRwl traveled with -Dwl . In MLCs between panel donors sharing zero, one, or two HLA-D-related B-cell specificities, significantly weaker MLC stimulation was observed with increasing compatibility, the median relative responses being 100, 52, and 17 percent, respectively. I t is concluded that B cell-specificities HLA-DRwl-7 and WlA8 are probably coded for by HLA-D; they are excellent markers for the HLA-D determinants, which can thus be typed for by serological means; and serological typing for HLA-D has great value in predicting the outcome of MLCs.

Introduction

The mixed lymphocyte culture (MLC) interaction is thought to be an in vitro correlate of the initial events occurring in vivo following challenge by allogeneic cells. In the human, the strongest stimulation in MLC is caused by disparity at the H L A - D locus (Yunis and Amos 1971, Thorsby and Piazza 1975). The HLA-D determinants are definable in MLCs by HLA-D homozygous cell typing (HCT) and primed lymphocyte typing (PLT) techniques, both having insufficient discriminative power and both of which are time-consuming (120 and 40 hours, respectively; see Thorsby et al. 1977 for discussion).

Serological detection of HLA-D-associated antigens present on B cells and macrophages was first reported in 1975 (van Rood et al. 1975) and later confirmed in many laboratories including our own (Solheim et al. 1975, Albrechtsen et al. 1977a). In the recent Seventh International Histocompatibility Workshop (7w), seven HLA-D-related B-cell specificities, HLA-DRwl-7, were established, cor-

0093-7711/78/0006/0091/$02.00

92 D. Albrechtsen et al.

responding to the H L A - D w l - 7 1 de te rminants in addi t ion to the less well-defined specificity W I A 8 , cor responding to D w 8 (Bodmer e t al . 1978). I t is the pu rpose o f this c o m m u n i c a t i o n to repor t the defini t ion o f these B-oeil specificities in our l abora to ry . Fur ther , results will be presen ted demons t r a t ing that the M L C - a c t i v a t i n g H L A - D de te rminan t s t a n n o w confident ly be typed for by serological methods , and tha t such typ ing has great value in predic t ing the o u t c o m e of M L C s .

Materials and Methods

Panel Cell Donors. These were 95 selected healthy unrelated Caucasian mernbers of the hospital staff and relatives of transplant patients, including 10 HLA-D-homozygous typing cell donors.

Farnilies. Five families seleeted for an HLA recombination event were investigated.

HLA-Identical Siblings. The siblings were relatives of transplant patients investigated as part of out kidney transplant program.

Peripheral Blood Mononuclear (PBM) Cells. PBM cells were separated from defibrinated blood by Ficoll-Isopaque flotation (Lyrnphoprep, Nyegaard & Co., Oslo, Norway). Ail panel cells were used freshly prepared.

B Cell-Enriched Suspensions. These were prepared by E rosetting of PBM cdls foUowed by Ficoll- Isopaque flotafion. Approximately 1 • 107 PBM oeils resuspended in 3 ml rnedium (RPMI 1640 with glutamate, Gibco Bio-Cuk, Glasgow, Scotland) were mixed with 3 ml medium containing 1% AET (Sigma, St. Louis, Missouri)-treated sheep red blood cells (Pellegrino et al. 1976) and 40% foetal bovine serum (Gibco Bio-Cult, Glasgow, Scotland) and centrifuged 200 G for 5 minutes at 20~ The pellet was gently resuspended, layered over Ficoll-Isopaque, and centrifuged 800 G for 20 minutes, leaving nonrosetting cells at the interface. These were 50-60% B cells [being stained by FITC-labeled F(ab'): fragments of rabbit anti-hurnan Ig in fluorescence microscopy] and 20-30% macrophages (as evidenced by latex particle ingestion; Albrechtsen 1977). From 70 to 100% of these cells were reactive in complernent-dependent cytotoxicity (CDC) tests with an anti-human B cell- (and macrophage-) specific xenoantiserum (serum p 23.30, kindly supplied by Dr. J. Strominger, Boston, Massachusetts) (Humphreys et al. 1976, Albrechtsen 1977).

HLA-A, -B, -C Typing. Typing was performed on PBM ceUs in CDC tests (Albrechtsen et al. 1977a), using 54 highly selected typing sera defining 11, 17, and 5 antigens of the -A, -B, and -C series, respectively.

HLA-D Typing. Typing for the HLA-Dwl-92 speeiflcities was performed by the HCT technique reported previously, using typing oeils procured locally or received through exchange (Thorsby et al. 1975, Kaakinen et al. 1977). Further, most ofthe panel cell rnembers were also typed by the 7w typing cells. Generally, responding cells were said to carry a given HLA-D speeificity if more than half of the cells apparently typing for this specificity (as shown by our own correlation analysis) gave stabilized relative responses (Ryder et al. 1975) of less than 40-50% (Kaakinen et al. 1977). Previous reproducibility tests had revealed less than 4% discrepant typing results (Kaakinen et al. 1977).

B Cell Specific Antisera were 174 antisera received through the 7w exchange, including own locally produced antisera (Albrechtsen et al. 1977a) (serum 50w = 7w014, serum AH = 7w024, serum TH = 7w032 and serum TL = 7w058). Also included were rive blind negative and positive control sera and two blind duplicates ofthe same B oeil specitic antisera.

Serological Typing ofB-Cel l Antigens. Typing was performed using the standard 7w CDC technique. Briefly, 0.5 gl B cell-enriched suspension (approximately 1 • 103 cells in medinm) was incubated for 60

1 New nomenclature: LD 107 and LD 108 have been named Dw7 and Dw8 respecfively. Further, three additional specificities Dw9, Dw 10 and Dwl 1 were provisionally named, sec "WHO Nomenclature Report" in Bodmer et al. (1978). 2 Dw9 was previously called LD-OH in communications frorn this laboratory

Predictive Value in MLC of Serological HLA-D Typing 93

minutes with 0.5/al antiserum in wells of microtest plates (Hamax A/S, Moss, Norway) covered by paraffinum liquidum, then for an additional 120 minutes with 2.5/al rabbit complement (received through the 7w from Dr. Kissmayer-Nielsen, Arhus), and finaUy for approximately 45 minutes with 0.5/al 1.8% trypan blue before cytotoxicity was scored. Ail incubations were performed at 20~ Cyto- toxicity exceeding 30% was scored as a positive result. The B-cell xenoantiserum (p. 23.30) and a multispeeific HLA antiserum (from a multitransfused patient) were included as positive controls, and pooled normal serum from healthy males was used as negative control. The baekground cytotoxicity was generally less than 10%. Reproducibility experiments included in the 7w (blind duplicate testings by the same antisera) gave correlation coefficients of 0.67 and 0.69 in our laboratory and blind positive and negative control sera were correetly scored in ail but four donors.

MLCs. MLCs between selected panel cell donors as well as between HLA-identicai siblings were performed as previously described (Thorsby et al. 1975). Briefly, 5 • 10 g responding PBM cells and 5 • 104 X-irradiated (2000 rad) stimulating PBM cells were cultured in 0.15 ml medium supplemented with 20% normal serum and antibiotics in round-bottomed wells of Cooke microtiter plates. The cells were harvested on glass fiber sheets after 120 hours, after having been labeled for 24 hours with 3H- thymidine, and counted by liquid scintillation. MLC results were expressed as relative responses (RR), calculated from median cpm of triplicate cultures according to the formula RR = ABx - AAx/ AYx - AAx, where A is responding oeils, B is stimulating eells, and Y is a pool of eontrol stimulating cells.

R e s u l t s

Association between B-Cell Antigens and HLA-D: Clusters o f B Cell-Specific Sera Strongly A ssociated with HLA-D. A p p r o x i m a t e l y 80 o f the B cell-specific sera tested reac ted with panel cells in a pa t te rn significantly ( p < 0.05) assoc ia ted with one or more H L A - D determinants . A n u m b e r o f sera fo rming well-defined clusters h a d a react iv i ty s t rongly associa ted ( p < 0.001) wi th the H L A - D de te rminan ts D w l - 5 and D w 7 - 8 , as is shown in Tab le 1. Se rum associa t ions with H L A - D w 6 were less significant ( p < 0.01). The clusters were provis iona l ly des ignated " I a " l - 8 , each cluster apparen t ly defining a separa te H L A - D - r e l a t e d B-cell specificity. N o sera had react iv i ty significantly assoc ia ted with D w 9 (identified by 7w cells 550 and 563), D w l 1 (identified by 7w ceU 566), o r L D - R E (Grosse -Wi lde et al. 1977; identified by 7w cell 567).

T a b l e 1. Clusters of B Cell-Specific Sera Strongly Associated with HLA-D

Cluster (and level value) a HLA-D 7w "Ia" Serum Number (and r value) ~ Association

"Ia"l (0.48) Dwl 152 (0.93) 128 (0.86) 120 (0.70) 009 (0.62) 042 (0.55)

"Ia"2 (0.38) Dw2 016 (0.90) 021 (0.87) 015 (0.78) 018 (0.73)

"Ia"3 (0.37) Dw3 024 (0.94) 028 (0.88) 180 (0.70) 133 (0.67)

"Ia"4 (0.70) Dw4 106 (0.46) 129 (0.46) "Ia"5 (0.70) Dw5 155 (0.84) 074 (0.38) "Ia"6 (0.75) Dw6 046 (0.30) 157 (0.29) "Ia"7 (0.28) Dw7 054 (0.80) 077 (0.79)

073 (0.71) 072 (0.69) "Ia"8 (0.52) Dw8 058 (0.77) 092 (0.62)

017 (0.82) 013 (0.80) 162 (0.72) 014 (0.70) 035 (0.75) 034 (0.74) 026 (0.65) 038 (0.40) 059 (0.35)

150 (0.25) 051 (0.76) 069 (0.69)

052 (0.72)

a Cluster level value = 1 -- r, where r i s the lowest correlation coefficient between any two sera included in the cluster (sec Bodmer et al. 1978) b Correlation coefficient to the given HLA-D specificity, r = ~ X 2 calculated with Yates correction. AI1 r values are signh�9 at 0.1% level, except r values for sera 046, 157, and 150, whieh are signifieant at 1% level


HLA-D ond

�9 Presence of HLA-D determinQnt c L 4 I " ) I "~ I l 1

5

10!

15

2s

25

30

35

z,0

Z,S

50

55

6C

6E

7C

HLA-DR PHENOTYPE DISTRIBUTION AMONG 72 UNRELATED NORWEGIANS

�9 Presence of HLA-DRQg ~ Doubtful

} 3 } ~. ) ~ ) 6 1 7 I a ]'N~,y

I I [ . I I I(OH)~ I(EI)

I l

I ™

Q

�9 Q

I,

Il

mi I!

I � 9

I � 9

I � 9

I �9 I � 9

I

I

Fig. 1. HLA-D and -DR specificities in panel cell donors

Association between B-CeE Specificities and HLA-D: Serological Typing of HLA-D. Each panel ceU donor was assigned B-cell specificities according t �9 the following criteria: HLA-DRwl, 2, 3, 4, and 7 were assigned if positivity was recorded with at least two of the three re�9 strongly HLA-D-associated sera within each corresponding cluster. DRw5 was assigned on the basis of positivity with �9 serum only (7w155), while DRw6 and WIA8 were assigned if both of two sera were positive (7w046-157 and 7w058-092, respectively).

A very strong association (p < 0.001) between the specificities DRwl-5 , 7, WIA8, and the corresponding HLA-D determinants was observed, as depicted in Figure 1 and recorded in Table 2. The less well-defined B-cell specificity DRw6 was significantly, though not s�9 strongly, associated with HLA-Dw6. Most discrepancies involved DRw4 and 6, where B-cell typing indicated the presence of these specificities without the corresponding Dw specificities, i.e., B-ceU typing was


Table 2. Serological Typing of HLA-D in 95 Cell Donors Using 20 Selected B Cell-Specific 7w Sera

HLA-D B-Cell + + + - - + - - r Specificity

Dwl D R w l 13 1 0 81 0.91 a Dw2 DRw2 22 1 1 71 0.91 a Dw3 DRw3 21 2 0 72 0.91 a Dw4 DRw4 13 1 11 70 0.61 a Dw5 DRw5 6 1 0 88 0.84 a Dw6 DRw6 12 4 13 66 0.47 a Dw7 DRw7 13 0 I 81 0.91 a Dw8 WIA8 11 1 1 82 0.87 a

a p < 0.001

broader. Only four cell donors were assigned more than two B-cell specificities, and DRw6 was involved in ail these (which were triplets).

Family Studies. The rive HLA recombinant families studied included one -A/C, one - A/B, two -A/D, and one -B/D recombinant child(ren). In these families, B-cell specificities HLA-DRwl--4, 6-7, and WlA8 (DRw5 was not present) traveled with the HLA-B-D chromosomal region; in the -B/D recombination, DRwl followed HLA-Dwl.

Influence of B-Cell Specificity Matching on the Outcome of MLCs. Among panel cell donors being assigned two HLA-DR specificities, 27 donors were selected and divided into nine groups so that the donors within each group were identical for both B-cell specificities assigned. The phenotypes involved were DRwl/2 , DRwl/3 , DRwl/7, DRw2/3, DRw2/6, DRw3/4, DRw3/6, DRw3/7, and DRw3/WIA8. Ail individuals included were also shown by HCT to carry the corresponding HLA-D specificity. By performing MLCs in a checkerboard fashion among these donors, relative responses were obtained in 80, 96, and 48 combinations involving disparity for two, one, and zero DR antigens between stimulating and responding cell donors. Results of part of one experiment are given as raw cpm _+ SE (and RR) in Table 3. RRs from ail combinations tested are plotted in Figure 2. Generally, RRs in combinations involving a disparity of two B-cell specificities were higher (median = 100 percent RR) than in those involving only one disparate B-cell specificity (median = 52 percent RR). In ail these combinations, the RR always exceeded 20 percent. In contrast, with a few exceptions, very low RRs (median = 17 percent RR) were observed in DR-identical unrelated combinations.

However, even lower RRs (median = one percent RR) were observed between HLA-identical siblings (Fig. 2, right). To evaluate whether this difference could be accounted for by stimulation caused by HLA-A and/or -B disparity in the unrelated DR-identical combinations, RRs in these combinations were plotted in Figure 3 according to the HLA-A/B incompatibility (stimulating cell donor expressing antigens hOt possessed by responding cell donor). The median RR was slightly higher (median = 20.5 percent) in MLC combinations incompatible for both HLA-A and - B than in those compatible for HLA-A (median = 16 percent) or HLA-B (median = 11.5 percent), but the differences were not significant by Wilcoxon rank sum testing. Unfortunately, no DR-identical HLA-A- and -B-compatible unrelated combinations were available for study.

Tab

le 3

. S

tim

ulat

ion

in M

ixed

Lym

phoc

yte

Cul

ture

s (M

LC

s) b

etw

een

Indi

vidu

als

Sha

ring

0,

1, a

nd 2

HL

A-D

R S

peci

fici

ties

Sti

mul

atin

g C

ell D

on

oP

Res

p. C

ell D

onor

M

.S (

1/7)

.T

(1/

7)

A.B

(3/

8)

T.M

(3/

8)

K.M

(2/

3)

S.G

(2/

3)

Ref

eren

ce b

M.S

(1/

7)

4196

_+

1467

c 15

,046

_+

929

29,3

46 •

28

58

32,5

02 _

+ 10

06

28,1

98 _

+ 30

81

0 42

97

10

.._.88

92

A.T

(1/

7)

8378

_+

102

308

• 75

18

,188

+_

366

19,8

23 +

188

4 19

,808

+ 7

09

37

0 83

91

91

A

.B (

3/8)

12

,-'69

4 +

2288

11

,'647

•

117

--

192

_+ 1

07

"-37

9 +

565

"-51

56 +

480

13

3 12

2 0

2 53

T

.M (

3/8)

15

,"-50

1 •

818

12,-

"613

+ 11

46

-]84

9 •

199

~7

8 •

27

9 "-

7111

•

79

121

98

11

O

53

K.M

. (2

/3)

18,--

-880

f 13

42

13,-

-698

• 17

54

8815

•

1398

92

16 •

17

42

264

• 47

6 11

5 83

52

55

0

S.G

(2/

3)

22,--

-461

• 23

6 18

,514

•

414

11,1

91

f 62

2 10

,200

•

894

1697

•

338

128

104

61

55

5

31,1

96 +

214

6 10

4 25

,557

+_

139

117 56

71 •

81

1 58

72

24 +

_ 78

4 57

12

92 +

305

6 89

8 +

694

0

30,0

22 +

614

21,7

17 +

13

43

9565

•

1284

12,8

17 +

435

16,3

71

+ 65

9

17.6

92 _

+ 14

98

a H

LA

-DR

(or

WlA

) sp

ecif

icit

y gi

ven

in p

aren

thes

es

b S

tim

ulat

ing

cell

s po

oled

fro

m a

il te

sted

don

ors,

use

d as

ref

eren

ce fo

r ca

lcul

atin

g R

R

c m

ean

cpm

i

S.E

. a

Rel

ativ

e re

spon

se (

RR

) =

AB

x --

AA

x/A

Yx

- A

Ax,

whe

re A

is

resp

ondi

ng c

ell,

B s

tim

ulat

ing

cell

, and

Y r

efer

ence

cel

l

> ,q

™


% RR' 140-

120

100

80

6O

40

20.

0

-201

,,.': OO

OO

'O

�9 o

$"

I

n=80 I'~ � 9 I

Two antigens

HLA-DR

~ 0

o o o

Oo~. ~o

O O o 5

-'._-.,.4 ™

One antigen

HLA-DR DISPARITY v.

MLC RESPONSE

~ �9

?~ n = 48 "I~'IIT-*~% ~ - ~O O0

�8 : 17 *I, RR ,,o:,~t ~o

No i antigen

DISPARITY

�9 n =100 * ™ :1 * / ,RR

HLA IDENT SIBS

Fig. 2. Relative responses in mixed lymphocyte cultures between unrelated cell donors disparate for 2, I, and zero HLA-DR specificities, and between HLA-identical siblings

Discussion

We have previously reported the serological identification of five B-cell specificities, all strongly correlated to HLA-D (Albrechtsen et al. 1977a). As a result ofthe serum exchange organized as part of the Seventh International Histocompatibility Work- shop, we were able to define eight B-oeil specificities strongly associated with the HLA-D determinants Dwl-8, which, in our North European population, cover a gene frequency of 0.76 (Kaakinen et aL 1977). Their strong association with HLA- D, their apparent mutually exclusive expression in the cell donor panel (four triplets out of 95 individuals tested), and their inheritance in the recombinant families studied strongly indicate that the presently defined B-cell specificities are coded for by one lotus, and that this loeus is probably identical to H L A - D .

98 D. Albreehtsen et al.

That major MLC-stimulating specificities were indeed identified by the selected B-oeil antisera was verified in MLCs between donors sharing zero, one, and two HLA-DR specificities. These experiments revealed a generally weaker MLC stimulation with increasing DR compatibility. Furthermore, antisera directed against some of these DR speciticities are able to inhibit MLC stimulation (van Leeuwen et al. 1973, Albrechtsen et aL 1977b). Whether the same determinants are responsible for T-cell activation (in MLC) and for triggering of antibody production is presently unknown, However, our resuks show that these B-oeil speciticities are excellent markers for HLA-D, and that their definition by presently available antisera makes it possible to type for HLA-D by serological means, as previously suggested (van Rood et al. 1975, Solheim et al. 1975, Albrechtsen et al. 1977a).

The stimulation observed in (the serologicaUy defined) HLA-D-identical unrelated MLC combinations was generally found tobe stronger than that in HLA- identical siblings. One explanation is that the serologicaUy defined HLA-DR specificities, as is the case for the MLC-activating HLA-D determinants, are not sufficiently defined to allow accurate typing of unrelated individuals. By contrast, HLA-identical siblings carry identical HLA-D determinants by descent (if no recombination has oceurred). Secondly, there may exist additional HLA-coded loti, giving rise to minor MLC-activating determinants, which may cause stimulation in HLA-D-identical unrelated, but again usually not in HLA-identical sibling combinations. To investigate whether the H L A - A and B loti (or regions) might code for such determinants, the material was subdivided according to HLA-A or -B compatibility (Fig. 3), but only slightly lower MLC responses were found in the HLA-A- or HLA-B-compatible combinations. However, the most informative combinations, being HLA-A- and -B- (as well as -D)-compatible are lacking. Previous experiments (Eijsvoogel et al. 1972, Thorsby et al. 1973) have indicated a second MLC locus, LD-2, in the vicinity of, or identical to, H L A - A . van Rood et al. (1977), in experiments similar to those reported above, found evidence for some MLC stimulation caused by HLA-A and -B disparity. In the mouse, several studies

Vo RR 100

8O

60.

z,0.

20,

MLC response between HLA - DR ,, identicol unreloted InfLuence of HLA-A or B compcttibitity

~ee eo �9 o~~ | ~

ooe �9 ~

oo~Oeo ~ ~ 1 7 6 �9

H L A - A ~ HLA-A HLA-B H L A - B ~ comp comp

Fig. 3. Relative responses in mixed lymphocyte cul tures between HLA-DR-identical unrelated cell donors compatible for either HLA-A or B, or i n compatible for botb HLA-A and B


have indicated that products of the H-2K and -D loci are also involved in M L C stimulation (see Klein 1975), and by using responding oeils from in vivo immunized volunteers, we have found that the H L A - A and -B antigens as such may also cause stimulation in human M L C (Bondevik and Thorsby 1974).

H L A - D typing by means o f the H C T method has previously been shown t o b e helpful in predicting the outcome of M L C s (JCrgensen et al. 1975, Thorsby et al. 1975). The predictive value o f serological typing o f H L A - D was found to be (at least) equally good. A weak M L C response of the recipient-versus-donor is generally thought to augur a good prognosis in clinical t ransplanta t ion (Cochrum et al. 1973, Solheim et al. 1977). However, previously available techniques have allowed only H L A - D matching t o b e possible in related donor t ransplantat ions. Serological typing for H L A - D and its predictive value in M L C now make it possible to match for H L A - D as well in unrelated combinations. Our findings indicate that a very low M L C activation is to be expected in unrelated combinat ions compat ible for H L A - A , -B, and -D. The influence on cadaveric kidney graft survuval of such an extended histo- compatibi l i ty matching, and whether this procedure m a y make it possible to select unrelated donors for clinical bone mar row transplantat ion, remain t o b e investigated.

Acknowledgments. We are grateful for the expert secretarial help of Ms. Elizabeth Garmann and Ms. Kristi Goyer. This work was supported by grants from the Norwegian Council for Science and the Humanities, the Norwegian Cancer Society, and Mr. Anders Jahre's Medical Research Fund.

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Klein, J.: Biology ofthe Mouse Histocompatibility-2 Complex. Springer-Verlag, New York, 1975 Pellegrino, M.A., Ferrone, S., and Theofilopoulos, A.N.: Isolation of human T and B lymphocytes by

rosette formation with 2-aminoethylisothiouronium bromide (AET)-treated sheep red blood cells and with monkey red blood cells. J. IrnmunoL Meth. 11:273-279, 1976


Ryder, L.B., Thomsen, M., Platz, P., and Svejgaard, A.: Data reduction in LD-typing. In F. Kissmeyer- Nielsen (ed.): Histocompatibility Testing 1975, pp. 557-562. Munksgaard, Copenhagen, 1975

Solheim, B.G., Bratlie, A., Winther, N., and Thorsby, E.: LD typing with antisera produced by planned immunization. In F. Kissmayer-Nielsen (ed.): Histoeompatibility Testing 1975, pp. 713-718. Munksgaard, Copenhagen, 1975

Solheim, B.G., Engebretsen, T.E., Flatmark, A., Jervell, J., Enger, E., and Thorsby, E.: The influence of HLA-A, -B, -C and -D matching on kidney graft survival. Scand. J. Urol. Nephrol. (SuppL) 42:28- 31, 1977

Thorsby, E. and Piazza, A.: Joint report from the Sixth International Histocompatibility Workshop Conference. II. Typing for HLA-D (LD1 or MLC) determinants. In F. Kissmeyer-Nielsen (ed.): Histocompatibility Testing 1975, pp. 414-458. Munksgaard, Copenhagen, 1975

Thorsby, E., Hirschberg, H., and Helgesen, A.: A second locus determining human MLC response. Separate lymphocyte populations recognize the products of each different MLC locus allele in allogeneic combinations. Transplant. Proc. 5:1523-1527, 1973

Thorsby, E., Bratlie, A., Helgesen, A., Rankin, B., Solheim, B.G., Kaakinen, A., and M611er, E.: Human MLC activating determinants. In F. Kissmeyer-Nielsen (ed.): Histocompatibility Testing 1975, pp. 502-508. Munksgaard, Copenhagen, 1975

Thorsby, E., Albreehtsen, D., Hirschberg, H,, Kaakinen, A., and Solheim, B.G.: MLC-activating HLA- D determinants: identification, tissue distribution and significance. Transplant. Proc. 9:393-400, 1977

van Leeuwen, A., Schuit, H.R.E., and van Rood, J.J.: Typing for MLC (LD) II. The selection of non- stimulator cells by MLC inhibition tests using SD-identical stimulator cells (MISIS) and fluorescence antibody studies. Transplant. Proc. 5:1539-1542, 1973

van Rood, J.J., van Leeuwen, A., Keuning, J.J., and Bluss› van Oud Alblas, A.: The serological recognition of the human MLC determinants using a modified cytotoxicity technique. Tissue Antigens 5:72-79, 1975

van Rood, J.J., van Leeuwen, A., Termijtelen, A., and Bradley, B.: The predictive value of MLA-D matching for the outcome ofthe MLC test. TissueAntigens 10:148, 1977

Yunis, E.J. and Amos, D.B.: Three closely linked genetic systems relevant to transplantation. Proc. Natl. Aead. Sci. U.S.A. 68:3031-3035, 1971

Received October 24, 1977

Documents

Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC)