Scand. J . Irnrnunol., Vol. 5 , Suppl. I, 1976.

TYPing for MLC Determinants Methods and Applications M. THOMSEN, L. P. RYDER & A. SVEJGAARD Tissue Typing Laboratory, Blood Grouping Department, State University Hospital, Copenhagen, Denmark Abstract. MLC typing of random individuals can be performed using a panel oi inactivated HLA-D homozygous cells. Eight different HLA-D spccificities are now internationally accepted. The evaluation of the results must take into account both the general responding capacity of the cells to be typed and the general stimulating capacity of the typing cells. An evaluation based on the 75th percentile is discussed in detail and some pitfalls are mentioned. Furthermore a description is given of primed lymphocyte typing (PLT), where cells primed in ordinary MLC cultures to one HLA-D determinant have the ability to respond in an accelerated way to similar HLA-D antigens when re-exposed to such cells in secondary cultures. In our experiments, an excellent correlation is found between these two ways of MLC typing provided that the cells used for priming are well characterized (i.e. HLA-D homozygous cells). Finally, some clinical applications of MLC typing are described, especially in connection with transplantation and association between HLA and various diseases.

When tissue is transplanted from one member of a species to another, it is usually rejected by immunological mechanisms because it carries histocompatibility antigens not present in the recipient. Each vertebrate species has one highly polymorphic system which is the strongest histocompatibility barrier within that species. In man, this major histocompatibility system has been named the HLA system (the first ( A ) Human Leucocyte antigen system discovered). Like its counterpart in other vertebrates, the HLA system controls various categories of antigens: (i) the so-called SD (serologically detectable) antigens, which are present on most types of cells and which are controlled by multiple alleles at at least three different loci (HLA-A, B, and C) ; (ii) the MLC ( = mixed lymphocyte culture) or LD (lymphocyte defined) antigens, which are the topic of this paper and which are primarily present on B lymphocytes and controlled by multiple alleles at at least one locus (HLA-D); (iii) the socalled Ia (immune-region associated) antigens, which are also primarily present on B lymphocytes, on which they can be recognized by

serological methods. In addition, major histocompatibility systems also contain genes which control various components of the complement cascade. In man, the properdin factor B genes (Bfand B s ) and the genes for the second and fourth component (C2 and C4) are located within the HLA system. Finally, major histocompatibility systems contain Ir ( = immune response) genes, which control the immune response to a variety of antigens. For detailed information on the HLA system, see references 1, 11, 29, 33. The mixed lymphocyte culture reaction is due to differences in MIX or HLA-D determinants on the B lymphocytes from the two individuals. These determinants are recognized by ‘receptors’ on T lymphocytes of individuals lacking the HLA-D antigens in question, and the subsequent blast transformation and cell proliferation can be measured by the incorporation of labelled thymidine in newly synthesized DNA. Within the last few years it has become possible to type individuals for a number of HLA-D determinants, which - like other HL.A

158 M. Thomsen, L. P . Ryder C A . Svejgaard THE MAJOR HISTOCOMPATIBILITY SYSTEM HLA IN MAN

D

B

C

A

I

I

-

I

ML

FO’JR

AJ

LA

Blood groups CHID0 and RODGER 0’ FOCIOI 0 polymarc%#sm

C2. and C L * l ? l i C B ’ ? i I,?

107

Fig. 1. HLA chromosome. Relationship between the HLA-A, B, C, and D loci on chromosome no. 6. The distance between the A and B loci is approximately 1 centimorgan. PGM3 is the locus for phosphoglucomutase-3. The lines below the chromosome indicate the approximate positions of the genes coding for the markers in question.

ONE-WAY MLC

Responding cell

/ \

Stimulating cell

tcell divisions

1

2

3

Fig. 3. Kinetics of MLC sponses.

5 time (days) ( 0 )and PLT ( 0 )re-

4

antigens - appear to be codominantly inherited. In one typing procedure, HLA-D homozygous ‘typing cells’ are used, in another more recently developed test, advantage is taken of the fact that lymphocytes which have been primed with a specific HLA-D determinant respond more rapidly to cells carrying this determinant than to cells lacking it. Below we shall briefly describe these two methods for HLA-D typing with special emphasis on the evaluation of the results. In addition, the present state of knowledge concerning the HLA-D polymorphism is reviewed and some applications of HLA-D typing are discussed.

MLC TYPING WITH HLA-D HOMOZYGOUS CELLS Principles Typing of HLA-D determinants with HLA-D homozygous cells was described independently by four different groups in 1973 (5, 16, 19, 36). The basis for the typing is the assumption that non-stimulation in the MLC reflects identity in the HLA-D determinants and converse-

Typing for MLC Determinants

159

Table I. The principle of MLC typing with HLA-D homozygous stimulating cells

HLA-D genotype Responder

Stimulator (typing cell)

alb ala

ala ala

Clb

ala

MLC response

Phenotype of responder

weak neg. pos.

a+ a+ a-

Letters a, b and c indicate HLA-D determinants.

ly that stimulation indicates differences between them. The proliferative response of the lymphocytes in the MLC is measured by the uptake of labelled thymidine into the DNA of the dividing cells and gives an estimate of the non-identity of the two cell populations. In the one-way MLC the cells from one of the cell donors are treated with mitomycin-C or Xirradiation, and these cells (the stimulator cells) are not able to divide or incorporate thymidine to a significant degree, but are still capable of inducing transformation of the other set of cells (the responder cells). If the stimulator cells are homozygous for the HLA-D determinant, they can be used for typing of HLA-D determinants. As shown in Table I, the cells to be typed can possess the HLA-D determinant of the typing cell in either a homozygous or heterozygous state or not at all. In the first two cases, we see a typing response, i.e. a proliferative response significantly lower than that of the cells not possessing the determinant typed for. Thus, by establishing a panel of HLA-D homozygous typing cells, it is possible to assign the HLA-D determinants of random individuals. Sources of typing cells An obvious source of typing cells is the children of consanguineous matings. In incestuous matings (father-daughter, mother-son or sibling-sibling) about 25% of the children will be HLA homozygous, and in children of first cousin marriages the chance of homozygosis is about 6% (Fig. 4). In inbred

Fig. 4. Pedigree of a first-cousin marriage offspring. Black areas symbolize one IILA haplotype inherited through the four generations.

populations in isolated areas there is also a good chance of finding HLA homozygotes, although it is difficult to give estimates. It is also possible to find HLA-D homozygous individuals in an outbred population. This is due to the fact that HLA-D polymorphism is limited, so that several of the alleles have gene frequencies of about S-lO%. If there were only 10 alleles, each with a gene frequency of lo%, 10% of the population would be potential typing cells, but this estimate is too high, as the number of alleles is greater and some of them are probably rare. In the search for outbred typing cells, there is an advantage in the fact that a linkage disequilibrium exists for the HLA region. This means that certain combinations of antigens

160

M.Thornsen, L. P . Ryder 6 A . Svejgaard

from the HLA-A, B, C and D series are found together more often than would be expected from the gene frequencies alone. For instance, strong linkage disequilibria are found between HLA-Bw35 and Dwl, HLA-B7 and Dw2, and HLA-88 and Dw3. Consequently, in individuals homozygous for a certain HLA-B antigen the chance of finding HLA-D homozygotes is considerably increased and many typing cells have been found in large MLC experiments between such individuals. Many typing cells have also been found incidentally through MLC studies in families, and as the critical MLC combinations for establishing HLA-D homozygosis are often easily at hand, random family studies can be useful in this respect. To prove the HLA-D homozygosis of a given individual, one of the following criteria must be fulfilled. a) The cell must not stimulate strongly any of the relatives in MLC if they share one of the HLA haplotypes with the potential typing cell. b) The cell must not respond towards or be stimulated by another HLA-D homozygous cell with the same specificity. c) The cell must 'type out' the same individuds, when used as a stimulator in MLC, as a proven HLA-D homozygous cell. As usual when biological factors are involved, no criteria are absolute, and this will be discussed later. Finally, some typing cells have emerged from studies of rare recessive diseases, where there is often a certain degree of consanguinity between the parents, although they are sometimes unaware of it. In particular, when the genes responsible are located close to the HLA complex (e.g. C2 deficiency), the chance of HLA homozygosis is, of course, significantly increased.

Methods The following methodological considerations are generalized to cover the different techniques employed in different laboratories. For detailed descriptions see reference 22. The blood sampling is done under sterile conditions and the coagulation is prevented

by addition of heparin, but other anticoagulants (e.g. ACD or EDTA) can also be used. However, defibrinated blood is also suitable. If the blood is not processed immediately, it is advisable to dilute it with an equal volume of tissue culture medium (e.g. RPMI 1640). It can then be stored or shipped at room temperature for 24 hours or more. Isolation of lymphocytes (mononucleas cells) is usually performed on a Ficoll-Isopaque gradient (2) but other sorts of gradients can also be used (metrizamide, dextran or albumin). The lymphocyte-rich layer is harvested and washed in medium or balanced salt solutions, often with the addition of 5-20% serum, which minimizes cell loss. The lymphocytes are counted and the cell concentration is adjusted to a fixed number of cells. Most laboratories employ 5 x 104 responding and 5-20 x 1 0 4 stimulating cells per culture. The stimulating cells are most easily inactivated by irradiation with X-rays, but this can also be done by treatment with mitocycin-C followed by thorough washing of the cells. The culture medium (RPMI 1640, TC199 or other 'rich' media) is supplemented with serum, usually 10-20%. The serum is usually a pool from non-transfused males to avoid possible interference from HLA antibodies. ABO blood group antibodies do not influence the results and the serum donors need not have the same blood group. The culture medium is buffered with HC03 and usually also with Hepes. Antibiotics are added to avoid possible microbial contamination. The cultures are usually set up in triplicate, either in culture plates or in plastic tubes, which are more medium-consuming, but perhaps yield a more stable and flexible system. The cultures are incubated at 37"C, 5% COZ in humidified air for 4-7 days. Before termination of the cultures, labelled (3H or *4C) thymidine is added to the cultures for a defined period of time (usually 24 hours). The cell proliferation is stopped by placing the cultures in 4"C, the cells are harvested on glassfibre filtres and the supernatant is removed by washing with distilled water.

Typing for MLC Determinnnts

161

Incremental CPM

7000 6000

Fig. 5. Illustration of the effect of the data processing. a. For each responder preparation (cf. Table 11) all the responses (actual cpm minus control cpm) elicited by the 15 stimulators are shown. The arrows indicate the 75th percentiles, i.e. the values selected to be the 100% references for the Relative Responses (RR) the 11th smallest in the samples of 15 values (see text).

i

5000 I000 3000

2000 1000

i

0

. : :

!' . 7

s

1

1 2 3 L

.

T

11

.

' 1

1

1

1

1

1

1

5 .

, 1

*

: . 1

1

1

1

1

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Responder

6. For each stimulator preparation all the 20 RR are plotted. It appears that some stimulators are generally low and others generally high. In order to stabilize these trends, the 75th percentile, i.e. the 16th smallest value among the 20 figures for each stimulator, is selected (arrows) as a 100% reference for the Stabilized Relative Responses (SRR) . To the right is shown a histogram of all the RR in this experiment.

c. The data shown in the same way as in b, but after the stabilization. The scoring code is indicated to the right of the diagram. Some of the stimulator preparations apparently do not allow a clear distinction between typing and nontyping in spite of this data processing. To the right is shown a histogram of all the SRR in this experiment. Note that the 'shoulder' to the left is more pronounced than in b.

. .

t

t

' ,

. . . . . .

No

I110 160 140

120 loo

80 60

Number

1140 120 loo

80 60 40 20 0 Stimulator No

Numbe I

162

M.Thomsen, L. P . Ryder 6A . Svejgaard

The incorporation of labelled thymidine into each culture is determined by placing the filtres in scintillation vials with scintillation fluid and counting them in a ,@-counter.The background activity is subtracted from the actual counts, and the counting is performed until a certain accuracy (often 5 % ) is obtained.

help. Nevertheless, no method, however sophisticated, is likely to give completely consistent results in the present state of knowledge and technology concerning the HLA-D typing. The raw material for the analyst is the single cpm, as obtained from the p counter, corresponding to each individual culture. In order to reduce the influence of technical and purely statistical variations, the individual MLC comEvaluation binations are usually set up e.g. in triplicate, The results of MLC-typing experiments are each combination thus giving rise to three rarely completely negative, as they are in MLC single cpm. Without losing very much informatests between HLA-identical siblings. The tion, a triple cpm can be represented by its most important reasons for the weak reactivity mean or its median. The median has the instead of non-reactivity of the ‘typing response’ advantage that it is not influenced by single outliers, which are not uncommon and are are: 1. Although the stimulating cells cannot usually due to technical errors. A conceptually and technically convenient divide, they do recognize the foreign HLAD determinants of the responder cells and way of setting up an MLC experiment is to release blastogenic factor, which induces regard each MLC combination as a ‘treatment’ some proliferation among the responder (by the stimulating cells) of the given responder. Thus the outcome of an individual comcells (back stimulation). 2. The HLA-D determinants of unrelated bination can be considered the joint result of individuals sometimes show similarity rather the general responding capacity of the responder, the general stimulating capacity of the than complete identity (cross-reactivity) . 3. Incompatibility of other factors in the HLA stimulator, and the specific interaction between region may theoretically give rise to weak the responder and stimulator. A particular responder or stimulator preparation is fairly responses. In addition to these HLA-relevant variables, often seen to give a whole array of low renoise is introduced into the system by general sponses. A likely technical explanation could variations at several levels: day-to-day, within be variations in the concentrations of viable the experiment, biological and technical, etc. cells. Occasional combinations of such low This makes it essential to consider the entire responders and low stimulators give an even experiment and not just the individual MLC lower outcome, which invalidates the conclucombinations. In general, it seems as if the sions based solely on the individual cpm. In order to reduce the influence of varialarger experiments give the most reliable tions in these general capabilities, we must conclusions. During the Sixth Histocompatibility Work- relate the individual responses to the general shop three schemes of evaluation were em- performance of the actual responder and ployed: the clustering method (21), a technique stimulator. Consider first a given responder and the based on the analysis of variance (20) and a array of responses elicited by the various ‘nonparametric’ approach ( 2 4 , 32). The first two methods have some desirable stimulators. Table IIa shows the individual properties in a statistical sense, but require median cpm from a routine MLC typing rather elaborate and time-consuming data experiment, where e.g. responder 1 consistently processing. The last method, which can be gives low responses, while responder 10 gives accommodated to a mini-computer or a large high responses (Fig. 5a). Many measures of desk-top calculator, has been used routinely the general level for a responder can be by us and has proved to be of considerable conceived, e.g. the mean, the highest value, the

163

Typing for MLC Determinants

median, etc. In fact, we were merely interested in obtaining a value reasonably representative for a positive MLC response of this responder in this experiment. The mean would be rather sensitive to outliers, the highest value is the outlier if any, and the median is sensitive to the occurrence of many typing responses. During the Sixth Histocompatibility Workshop we came to the conclusion that a reasonable value could be the 75th percentile* of the array. This was based on extensive histogramming for each responder, which showed that the (presumably) positive MLC responses usually clustered above the medians (Fig. 5b). Taking the 75th percentile as a representative positive MLC reaction for the given responder, the whole array of its responses are expressed as a percentage of this, after subtraction of the autostimulated control values, and this gives the relative responses (Table IIb). Considering next a given stimulator and the array of relative responses elicited by it (the columns in Table IIb), it appears that e.g. stimulator 12 gives a column of generally low values while stimulator 3 has a high level (Fig. 5b). To compensate for this variation we perform the same operation on the stimulator, i.e. we select the 75th percentile of the responses elicited by a given stimulator and express the column of values as a percentage of this. The figures obtained in this way have been called the stabilized relative responses, and are shown in Table Ilc. There are two reasons for choosing the 75th percentile as a reference value for the stimulator stabilization as well: the arguments from the responder relativation can be repeated, and furthermore the result of the symmetry is that in a homogeneous experiment the stabilization references will cluster around the value of 100%.

* The 75th percentile of a sample is defined as the value below which 3/4 of the observed values are found (26). This is not always an unambiguous definition; our calculator selects in a sample of N figures the value with the rank: integer part of [3/4”+0.5]. However, in larger experiments it usually makes no difference whether this value or its neighbours are used.

The results are more easily reviewed if the figures are transformed into an adequate scoring system. Since the. assignment of an HLA-D type by MLC methodology is based on a low reaction, the scoring should by analogy be chosen with ‘+’ representing combinations with low responses, and ‘-’ representing high responses. Our choice was a graduated scoring from ’ + + + +’ to as indicated in Fig. 5c. An example of the stepwise calculations is given in the following. I-’

In the MLC experiment shown in Table IIa responder no. 1 (the first row) is a low responder with median cpm ranging from 561 to 1,770 (i.e. from 213 to 1,422 above the control level of 348, Fig. 5a). This array of cprn are arranged in order by magnitude and the 75th percentile is found; this is the cpm which approximately divides all the 15 responses into y 4 of responses that are low and ?,4i that are high. The rank number of this value is the integer part of (3/4*15+0.5), or of 11.75, i.e. number 11. For responder no. 1, the 11th lowest cpm is 1,336, and the correspondin,g increment is obtained by subtracting the control value (348): increment = 1,336 - 348 = 988. This value is selected as the 100% reference for all the other increment responses of responder no. 1. For example, stimulator no. 1 gives a relative response of

561-34Kx 1,336 - 348

100

= 22%.

The same operations are performed for the following responders and the results are shown in Table IIb. Next, look at Table IIb stimulatomise and take for example stimulator no. 12. The relative responses in this column are all less than 100 (actually they are all less than SO), so stimulator 1 2 is a generally low stimulator (Fig. 5b), and the 75th percentile (having the rank of 15 in this sample of size 20) has accordingly only the value 61. Re-expressing all the relative responses as percentages of this value we obtain the stabilized relative responses (SRR) ; for example, responder no. 4 gives an SRR value of 53 -X 61

100

-

87% with this stimulator. Performing

similar operations with all the stimulators, we get Table IIc. In Table IId the SRRs have been transformed into ‘+’ and ‘-’ scores. It is worth noting that the combination of the low responder no. 1 with the low stimulator no. 1 2 gives 548 cpm above control and an SRR of 91, which is not interpreted as a typing response, while, for example, responder no. 11 stimulated with no. 14 (both of which are intermediate) gives 618 cprn above control, but an SRR value of 19, which is (considered a clear typing response.

C

a b

3,341 2,070 431 404 486

605.1 605.2 606.1 606.2 607.1

607.2 608.1 608.2 609.1 609.2

11 12 13 14 15

16 17

18

2,147 758 1,101 2,437 2,195

2,619 1,939 1,618 563 474

1,812 3,126 2,240 658 5,030

1,032 890 2,483 2,666 1,234

2

2,509 3,893 2,321 2,589 3,060

3,723 2,860 2,695 2,464 4,165

4,282 3,315 2,981 2,582 3,543

3,001 5,456 2,789 3,519 3,953

1,922 4,408 913 1,497 6,088

1,770 3,645 3,543 2,510 2,920

4

1,890 4,819 3,726 4,885 6,297

672 3,889 3,597 4,101 2,588

3

= responder. Id. no = identification number of responder. The figures represent median cpm.

R

19 20

2,258 4,542 1,474 2,806 3,041

1,658 1,708 2,868 3,373 5,148

602.2 603.1 603.2 604.1 604.2

5

4

2 3

6 7 8 9 10

561C 2,565 2,935 2,504 310

600.1 600.2 601.1 601.2 602.1

n0.b

1

1

Ra Id.

Stim. no.:

Table IIa

2,773 2,985 1,925 1,969 3,179 3,007 2,709 2,629 2,794 3,359

3,273 1,616 2,088 2,564 2,754 2,006 5,673 2,470 3,567 3,583

1,590 1,220 1,705 1,490 2,860 2,705 5,233 920 1,416 1,488

2,756 2,425 1,692 2,287 3,157 2,731 4,517 2,676 2,565 4,667

1,833 3,753 1,638 1,618 5,485

982 4,142 3,496 4,037 3,982

2,548 2,742 2,407 3,214 5,338

578 2,961 2,780 3,999 2,518

1,245 3,294 3,487 1,799 2,035

8

1,336 4,304 2,338 3,139 1,964

7

1,280 1,661 3,189 3,229 1,826

6

1,437 3,237 4,137 1,685 1,649

5

2,785 5,046 2,202 2,682 4,171

3,119 3,487 2,144 2,473 3,810

513 3,528 3,297 4,323 3,169

1,356 3,382 4,173 2,548 2,396

9

3,172 5,120 2,870 3,467 3,676

4,293 3,929 2,437 2,518 3,512

2,015 4,063 3,777 4,544 5,791

600 3,097 3,930 4,232 2,208

10

1,800 4,893 2,705 3,426 3,618

4,298 2,987 725 473 399 2,871 5,884 2,600 3,925 5,472

2,151 855 652 2,002 2,625

2,343 2,336 3,325 4,322 5,779

769 3,262 4,114 4,415 540

13

2,275 2,133 1,584 495 349

1,081 3,073 1,902 892 4,847

1,142 2,962 3,621 3,761 5,343 2,894 1,869 2,380 2,218 2,647

896 460 2,249 2,485 1,305

12

1,266 2,787 2,020 3,071 1,758

11

2,886 5,077 1,202 1,916 1,869

1,008 1,112 2,400 1,910 2,847

2,092 2,399 2,895 3,750 4,801

1,322 1,423 2,922 3,885 1,957

14

4,473 6,953 3,987 4,274 4,556

4,711 3,467 3,406 2,750 4,027

3,216 4,352 4,908 4,661 7,590

1,431 3,733 3,622 5,144 3,882

15

169

114 257 105 248

390 58 348 95 197

87 514 414 203 1,332

348 110 82 900 366

CTR

2,886 5,233 2,676 3,467 3,953

3,723 2,987 2,400 2,473 3,512

2,015 4,063 3,496 4,322 5,779

1,336 3,382 3,622 3,885 2,208

100%

Table I1 a-d. Calculator output of a standard MLC typing experiment. Each cow represents one responder identified by the numbers given in the two first columns. Each column represents one stimulator. CTR = control values. Responders 1-4 have known H U - D types and were included as controls, while responders 5-20 were random blood donors. For further explanations see text.

F

%

5'

9

I ,

,2-+

!xi

'26

-2 P

s,

Q

3

%

A

o\

-

77 86 53 79 76

89 69 4 13 9

80 77 86

34

81

-3

54

22c 75 81

1

54

73 10 39 68

67 64 62 20 8

89 74 59 11 83

69 24 68 59 47

2

104 104 104 102 100

128 105 101

111

117

107 114 112

121

94

115 99 107 121

33

3

a R = responder. b Id. no. identification number of responder. c The figures represent relative responses.

16 17 18 19 20

15

14

607.2 608.1 608.2 609.1 609.2

605.1 605.2 606.1 606.2 607.1

11

12 13

602.2 603.1 603.2 604.1 604.2

600.1 600.2 601.1 601.2 602.1

6 7 8 9 10

5

4

3

1 2

Ra Id. n0.b

Stim. no.:

Table IIb

92 89 94 86 100 72 119

86 73 86 73 76

65

71 81

25 69 77 92 27

110 96 115 26 70

5

100 96 114 100 120

95 110 16 31 107

139

54

144 108 98

4

93 100 32 36 35

36 40 66 59 80

128 63 65 73 90

94 47 88 78 79

6

68 109 92 103 90

85 104 77

53

86

46 102 100 93 60

75 87

64

100 128

7

104 49 98 79 84

71 100 77 79 90

91 91 40 34 93

91 97 96 30 91

8

96 96 82 76 106

82 117 88 100 109

22 85 94 100 41

110

55

102 100 116

9

110 98 108 100 93

117 132 102 102 100

100 100 109 105 100

100

112

26 91 109

10

61 93 101 99 91

75 62 99 89 74

55 69 104 86 90

93 82 55 73 76

11

140

114

54 65

99 113 97

117 100 18 16 6

100 100

94

117 51

96 114 118 9

43

13

73 12 21

57 71 60 17 5

52 72 48 17 79

61 53 51

11

55

12

100 97 43 52 45

19 36 100 76 80

80 86 78

53

104

99 40 80 100 86

14

151 125 116

157 135

130 116 149 112 116

141

162 108 146 108

110 111 100 142 191

15

607.2 608.1 608.2 609.1 607.2

16 17 18 19 20

=

75

100

97 108 67

108 15 57 100 79

98 94 71 29 12

132 108 87 16 122

102 35 100 87 69

2

68

72 92 72 87 88

103 78 113 92 87

82 107 95 100 98

75 100 71 106 76 126

80 68 80 67 71

b Id. no.

106 114 36 41 40

41 45 75 67 72

75 86

111

145 72 74 83 103

27 73 81 78 28

98 67

107 54 100 89 70

6

88

117 101 121 28 74

5

74

93 87 106 92

88 102 15 29 97

133 100 70 50 128

4

3

29 102 87 74 106

108

114

responder. = identification number of responder. c The figures represent stabilized relativc rrsponses.

a R

111

605.1 605.2 606.1 606.2 607.1

11 12

13 14 15

102 42 100 97 108

602.2 603.1 603.2 604.1 604.2

6 7 8 9 10

86 5 16 11

27C 94 101 67 -4

1

80

Ra Id. n0.b 1 600.1 2 600.2 601.1 3 4 601.2 602.1 5

Stim. no.:

100% Ref:

Table IIc

112

68 107 72 103 70

105 85 70

53

77 107 82 84 76

100

77 78 43 37

77 104 103 32 97

8

73

86 53 85 104 77

46 102 100 73 60

100 128 64 75 87

7

100

65 100 107 106 78

101 70 99 92 85 74 94 80 74 104

81 67 107 76 80

108 80 115 86 98 107

74 94 92

122

59 74 112 93 97

100 88 59 78 81

11

93

92 92 100 97 92

23 84 100 103 72

10

109

22 83 92 98 40

100 98 113 54 108

7

1Q2

120 20 35 87 106

98 27 7

11.6

92

84 118 79 27 129

91 17 100 87 83

12

61

87 99 85 100 123

103 88 16 14 5

103 45 83 88 88

37 85 100 103 8

13

114

103 100 44 53 46

19 37 103 79 83

107 55 83 89 81

102 41 83 103 87

14

97

108 92 104 86 80

89 80 102 77 79

74 100 74 97

111

75 76 69 98 131

15

146

K

607.2 608.1 608.2 609.1 609.2

16 17 18 19 20

= responder.

15

-

-1

-

-

**** **** ****

-

-

-

*

-

****

-?

-

***C

1

3

108 Mad

**** to - as in Fig.

2

3

LK

2

MST

c

Graduated scoring from

b Id. no. = identification number of responder.

a

605.1 605.2 606.1 606.2 607.1

11

12 13 14

602.2 603.1 603.2 604.1 604.2

Id. nob: 600.1 600.2 601.1 601.2 602.1

6 7 8 9 10

2 3 4 5

1

Ra

Stim. no.:

Specificity, Dw: Initials:

Table IId 107

5c.

4

Mou

4

5

BB

1

6

Mette 7

6 Preb 8

107 SL

108 Mad 10

4 Priess

9 11

6 Preb 12

3 Lone

2

13

RR

1

14

Maja 15

Dw:

POOL Conclusion

168

M.Thomsen, L. P. Ryder & A . Suejgaavd

Table 111. The principle of primed lymphocyte typing (PLT) test Responder

Primary stimulator

Specificity of ‘memory’ cells

Secondary stimulators

Secondary response

alb

alc

‘anti-C

alc cld bid

pos. pos. neg. neg.

XlY

alb

clc

‘anti-C

alc

bid XlY

During the last International Histocompatibility Testing Workshop both this rather simple approach and the more complicated clustering analysis were used on all the MLC data obtained from 15 different laboratories and both methods proved equally effective in defining typing responses. Pitfalls As described above, the results of typing for HLA-D are not always unequivocal. Repeated experiments can give a more reliable HLA-D assignment. A main objection to the use of a negative response in the definition of HLA-D determinants is the possible occurrence of non-reactivity towards MLC determinants not possessed by the individual to be typed. This has been suspected in some cases and actually shown to be true in one case (31). During the Sixth Histocompatibility Workshop an individual repeatedly gave typing responses with homozygous cells representing four different specificities. Only two of these specificities were found in the parents and her children also showed normal reactivities towards the ‘extra’ HLA-D specificities. Accordingly, we were dealing with an individual lacking the response to specific MLC determinants not possessed by her and this typing response was not dominantly inherited. The deduction from a negative observation to a positive assignment of an HLA-D specificity is thus not always correct and it was actually

pos. neg. neg.

Phenotype of sec. stimulator

C+ C+ CCC+ C-

C-

shown by the PLT test described below that this individual only possessed the two HLA-D determinants that could be defined in the family, and not the ‘extra’ ones. PRIMED LYMPHOCYTE TEST (PLT) Studies by Cerottini et al. (3) and Hayry et al. (12) some years ago have already shown that the responding lymphocytes in an MLC elicit a rapid secondary response when they are restimulated with the original stimulators. In 1975 Bach and coworkers (27) suggested that this aproach could be used for HLA-D typing. The crucial question is whether factors other than HLA-D determinants can elicit rapid responses in the primed cells. This will be discussed in detail later. The primary culture is made in such a way that the responding cells recognize only one foreign HLA-D determinant on the stimulating cells. When the MLC reaction has subsided after 10-14 days and ‘memory cells’ have been formed, the cells can be frozen or used immediately for secondary cultures. A rapid response will then occur if the secondary stimulators carry the same HLA-D determinant as the one the responder cells are primed against. Soarces of typing cells

Theoretically, all haplo-identical combinations within a family can be used as source for

Typing for NLC Determinants Fig. 6. Comparison between classical MLC typing and PLT. The calculation of the responses is explained in the text. The HLA-D specificity and the initials of the homozygous cells used for priming are indicated at the bottom. Open and filled-in circles indicate responses obtained with secondary stimulators lacking and possessing, respectively, the HLA-D determinant in question. Crosses represent responses obtained with the individual apparently carrying 4 HLA-D determinants by classical MLC typing (see text). From reference no. 31. S = specific (Le. priming) cells, H = HLA-D homozygous cells.

. .

S 0.

Dw 1

primed cells. In a family two parents and one child could be the source of four primed combinations: If the parental haplotypes are a/b and c/d, and the child a/c, then, as seen in Table 111, the following primed cells would be available: ‘anti-a’, ‘anti-b’, ‘anti-c’ and anti-d‘. However, several experiments indicate that, although they may work within the family, such combinations may yield conflicting results in the random population. We have tried another approach in order to get more clearcut results. If the stimulating cell is a well-defined HLA-D homozygous typing cell, the responding cell (not carrying the same HLA-D determinant) will be specifically primed against this determinant. Due to the gene-dose effect, the priming would be expected to be stronger, and this seems to be the case. The responder can be any person not possessing the HLA-D determinant in question, but undoubtedly some combinations will show better discriminatory power than others. However, it is also possible that haplo-identical combinations may give sufficiently strong priming if two or more primings (preferably with different stimulators) are done.

ow 2

Dw 3

Dw 4

169

..

X

LD 107

LD108

Methods

Lymphocytes are separated as described above. The primary cultures are set up in large quantities to obtain sufficient numbers of cells for several experiments. In our system, 107 responder and 107 stimulator (irradiated by 2,000 rad) cells are incubated in loosely capped Falcon flasks in 30 ml medium RPMI 1640, supplemented with 15% inactivated ( 5 6 O , 30 min) human serum and antibiotics. The flasks are placed in an upright position at 37OC, 5 % C 0 2 in humidified air, for 10-14 days. The cells are then washed once in RPMI 1640, and the number of viable cells (judged by 0.5% eosin) is counted. The recovery of viable cells is usually around 150% of the initial number of responder cells. The primed cells are then set up in cultures with 50,000 primed (responder) cells and 200,000 stimulating cells per culture. Due to the rapid proliferation of the primed cells, it is not necessary to inactivate the stimulating cells. The cultures are incubated under culture conditions as indicated above, and 14C thymidine is added 6-14 hours before the termination of

170

M . Thomsen, L. P. Rydeu & A . Suejgaard

Table IV. HLA-D antigen and gene frequencies in Danes HLA-D determinant

Frequency

Dwl Dw2 Dw3 Dw4 Dw5 Dw6 107 108 ‘Blank’

18.9 23.6 21.0 17.2 8.8 18.0 9.6 8.9

(yo)

Gene frequency

.099 .I28 ,122 .091 .045 .094 ,049 ,046 .336

Associated HLA-B determinant Bw35 B7 B8 BwlS Bwl6 Bw16 B12 Bw40

Data from ref. 32.

the cultures, which takes place at 24 or 40 hours, respectively. The cells are then harvested and the radioactivity counted as described under ordinary MLC typing. The specificity of the reaction can be increased by repeated priming of the responder cells by the same stimulating cells or other cells of the same HLA-D specificity (8, 10, 28). In this way it is also possible to increase the number of primed cells, as these proliferate upon repeated primings. It is worth noting that the primed cells are fairly easy to freeze down by the usual techniques for freezing of lymphocytes (22) and that their responding capacity (in contrast to that of unprimed lymphocytes) does not seem to be significantly affected.

Euuluation of results Owing to the limited experience of PLT typing, the best way of evaluating the results is not quite clear. The use of raw cpm’s is not very practicable since the responding capacity can vary from one primed cell to another. In our experience, the best basis for the evaluation is to take the cpm of the specific combination as the 100% value. As in ordinary

MLC typing, the stimulating capacity of the cells to be typed also shows considerable variations and various ways of eliminating this by statistical methods are under evaluation. If all the stimulating cells give at least one typing response with the primed cells, the stimulator variability can be normalized by using this typing response as the 100% value, but if there are no typing reactions this cannot be done. Another possibility is to compensate for the level of nonstimulation by taking the mean of the supposedly non-reacting combination. However, the results we have obtained so far indicate that the discriminatory power of the PLT test is so good that very sophisticated methods of statistical treatment of the results may not be necessary. Fig. 6 shows the results from one experiment and it appears that they clearly fall into two groups without overlapping. In this experiment the combinations were chosen in such a way that every stimulating cell had at least one positive reaction. The responses have then been normalized for the responders and for the stimulators, using the maximal stimulation as the 100% reference value. Only a few studies have been done so far to correlate the typing for HLA-D determinants by homozygous test cells and by the PLT method. According to our experiments these methods yield identical results, provided the priming combinations are chosen with great care. The heterogeneity of the HLA-D determinants causes conflicting results if randomly chosen PLT combinations are used as typing reagents. But if the combinations only differ for well-defined HLA-D specificities, the PLT method has great potential for the typing of HLA-D determinants. Its quickness makes it clinically applicable in the situation of organ transplantation and its positive identification of HLA-D determinants makes it a theoretically more satisfying method of HLA-D identification. As mentioned before, specific MLC nonreactivity has been observed in certain persons, which could not be explained by the possession of the HLA-D determinants in question, and in such instances the PLT method is superior to the ordinary MLC typing by homozygous cells.

Typing for NLC Determinants

Determinants During the Sixth International Histocompatibility Workshop in 1975 considerable effort was put into the definition of HLA-D determinants by means of MLC typing with homozygous cells. From the results it was possible to outline eight different determinants, which were defined by two or more different homozygous test cells (14, 30). In addition some single cells seemed to type for HLA-D specificities of rarer frequency. The frequencies of the eight commonest specificities in Danes are shown in Table IV, from which it appears that the combined frequency of as yet unknown genes is still of considerable size; it can be estimated that more than 50% of the population possess one or more unknown HLA-D determinants. During 1976 and 1977 a new workshop will be held, to try and define further specificities. Although eight different specificities are now recognized, few of the typing cells are completely alike. Each specificity is defined by a ‘cluster’ of homozygous typing cells, which in a given responder-panel do not always give the same typing responses. For instance within the Dw5 specificity some persons in the responder panel gave a typing response with all the typing cells in this ‘cluster’, whereas others gave a typing response to one subgroup in the cluster and others again gave a typing response to another subgroup of typing cells. The basis for this heterogeneity is as yet poorly understood, and as long as the structure of the MLC determinants is not known, the reason can merely be speculated upon. A characteristic of the MLC determinants is that they are not (or only to a small degree) present on T lymphocytes and thus bear a resemblance to the so-called Ia antigens. These antigens can be detected by a serological technique with B cell-enriched suspensions (23) and it has been speculated that they are identical to the MLC determinants. So far, some results indicate a very dose relationship between Ia typing and MLC typing for certain Ia antigens, but the correlation is apparently not absolute. This indicates that the genetic loci determining the MLC determinants and

171

some of the Ia antigens are closely associated, but not identical. SOME APPLICATIONS OF HLA-D TYPING Naturally, the above methods are necessary tools in clarifying the genetics and biology of the HLA system and in histocompatibility testing. In addition, HLA-D typing is relevant in studying some associations between HLA and disease. The genetics of HLA-D determinants have been discussed above. The biological function of these determinants is still obscure (as is that of most other HLA factors). Being present only or primarily on some types of cells, e.g. B lymphocytes, monocytes, endothelial and epithelial cells, and sperm, they may be considered differentiation antigens which are perhaps involved in the cooperation between these and other cell types. However, many other possibilities exist, and it is not the purpose of this survey to discuss this complicated but potentially very exciting area of research. The MLC test has long been considered an in vitro parallel of the allotransplantation reaction in vivo. It represents the recognition phase in which immunocompetent T lymphocytes recognize the foreign alloantigens and are accordingly stimulated to divide. Moreover, some of the cells in the culture even develop into killer cells which specifically lyse cells sharing HLA factors with the stimulating cells inducing the blast transformation. It has become increasingly clear t hat while the HLA-D determinants are responsible for the induction of cell division, it is not these determinants, but the classical HLA antigens of the A, B, C, and possibly other series which are the targets of the killer cells ( 6 ) . However, killer cells are unlikely to develop unless there has been some degree of cell division. Accordingly, it seems reasonable to assume that if MLC (HLAD) compatibility can be achieved, the chances of graft rejection should decrease. The first question to be answered concerns the possibility of selecting MLC negative combinations from a group of HLA-D typed individuals. Fig. 7 shows the results obtained

172 M . Thornsen, L. P . Ryder 6A . Svejgiiard SRR ( %)

.-

1-

!-

f. ...

i : 1

I

I

0

1

2

No incomp.

Fig. 7. Predictive value of MLC typing for the MLC reaction between unrelated individuals. 0 indicates combinations carrying the same two HLA-D determinants, 1 combinations differing for one HLA-D determinant, and 2 combinations differing by two. The experiments included 14 unrelated individuals typed for 8 different MLC determinants.

in a number of one-way MLC tests between various unrelated individuals sharing two, one, and no HLA-D determinants, respectively. The results in the three groups are clearly different, although there are some exceptions: stimulation was seen in some of the combinations sharing two HLA-D antigens. Possible explanations for these exceptions may be either false HLA-D typing (as discussed above, HLA-D typing is not always unequivocal), minor differences within an HLA-D cluster, or the existence of still unknown MLC determinants. Nevertheless, the generally low responsiveness seen in the group with two shared HLA-D determinants has also been found by others (15, 17, 34) and as HLA-D typing becomes more refined, an even better prediction

of MLC non-response or low response will be possible. The crucial point is, of course, whether MLC non-response is predictable for a successful transplantation. The very low rejection rate of kidneys from HLA identical and thus MLC negative siblings has long been an established fact, and there is now increasing evidence that MLC non-response or low response between the recipient and the donor is a favourable prognostic sign in cadaver kidney transplantation as well (7, 35, and our own unpublished data). Technically, bone-marrow transplantation is a fairly simple procedure, but immunologically it is probably the most difficult of all transplantations. Until recently, successful grafting without lethal graft-versus-host disease was only seen with bone marrow from HLA identical siblings. However, a few cases of successful bone-marrow transplantations have now been achieved with HLA-D compatible but HLA-A, B, and/or C incompatible donors ( 4 , 9, 18). Thus, the evidence is fairly good that HLA-D compatibility is one of the most crucial points in histocompatibility testing. A completely different field of research in which HLA-D typing has proved fruitful relates to the associations which have been observed between a variety of diseases and some HLA factors. The first studies of HLA and disease concerned only the HLA-A and B antigens, and most of the associations found are strongest with HLA-B antigens. Owing to the pronounced linkage disequilibrium within the HLA system, it is possible that some of these associations might be ‘secondary’ to a ‘primary’ association with other HLA markers associated with the HLA-B antigens in question. As there is a particularly strong association between HLA-B and D antigens, it was obviously of interest to study the latter in these diseases. Multiple sclerosis shows a rather weak association with HLA-B7 and now it has been shown that this association is due to a primarily increased frequency of the HLA-Dw2 antigen in this disease: About 60% of the patients possess this antigen as compared to only about

Typing for M L C Determinants

173

Table V. Some associations between HLA and disease Disease

HLA-B Antigen

Multiple sclerosis Insulin-dependent diabetes Idiopathic Addison's disease Graves' disease Dermatitis herpetiformis Subacute thyroiditis

IILA-D Relative risk

Antigen

-

Relative risk

B7 B8 Bwl5

1.63 2.4 2.5

Dw2 Dw3 Dw4

6.9 4.5 3.7

B8 B8

7.0 2.5

Dw3 Dw3

10.5 4.0

B8

18.2

Dw3

50.1

Bw35

16.6

Dwl

4.3

The relative risk indicates how many times more often the disease occurs in individuals carrying the antigen as compared to those lacking it. Data from references 13, 32, and Thomsen et al., unpublished.

20% of normal individuals (13). Analogously, some other disorders are apparently more strongly associated with HLA-D antigens than with HLA-B antigens (Table V) while still others (e.g. ankylosing spondylitis (25) and subacute thyroiditis) are not. The reason for the associations between HLA and disease is still unknown, but one of the presently most favoured explanations suggests that they are due to specific immune response (Ir) determinants which confer an abnormal immune reaction which in turn leads to the disease. In mice, the Ir genes within the H-2 system are situated very close to those controlling MLC determinants, and as there is a pronounced homology between H-2 and HLA, it is thought that the HLA-D determinants are also close to human Ir genes, which could explain the strong association with HLA-D for some diseases. However, no human Ir determinant has yet been identified within the HLA system because immune responsiveness is much more difficult to study in man than in laboratory animals.

ACKNOWLEDGEMENTS This study was aided by grants from the Danish Medical Research Council, the Danish Cancer Society, and the Danish Blood Donor Foundation.

REFERENCES 1. Bodmer, W. F. Evolutionary significance of the HL-A system. Nature (Land.) 237, 139, 1972. 2. Boyum, A. Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, Suppl. 97, 1, 1968. 3. Cerottini, J.-C., McDonald, H.R. & Brunner, K.T. Formation of cytotoxic lymphocytes in mixed leucocyte culture systems: Introduction. Transplant. Proc. 5 , 391, 1973. 4. Dupont, B., Andersen, V., Ernst, P., Faber, V., Good, R. A., Hansen, G. S., Henriksen, K., Jensen, K., Juhl, F., Killmann, S. A., Koch, C.,Muller-Berat, N., Park, B. H., Svejgaard, A., Thomsen, M. & Wiik, A. Immunological reconstitution in severe combined immunodeficiency with HL-A incompatible bone marrow graft. Transplant. Proc. 5, 905, 1973. 5. Dupont, B., Jersild, C., Hansen, G. S., Nielsen, L. Staub, Thomsen, M. & Svejgaard, A. Typing for MLC determinants by means of LD-homozygote and LD-heterozygote test cells. Transplant. Proc. S, 1543, 1973. 6. Eijsvoogel, V. P., Bois, M. J. G. J. du, Melief, C. J. M., Groot-Kooy, M. L. de, Koening, C., Rood, J. J. van, Leeuwen, A. van, Toit, E. du & Schellekens, P. T. A. Position of a locus determining mixed lymphocyte reaction (MLR) distinct from the known HL-A loci, and its relation to cell-mediated lympholysis (CML) . In Histocompatibility Testing, Munksgaard, Copenhagen, 1972, pp. 501-508. 7. Festenstein, H., Sachs, J. A., Paris, A. M. I., Pegrum, C. D. & Moorhead, J. F. Influence of HLA and blood-transfusion on outcome of 502 London transplant group renal-graft patients. Lancet i, 157, 1976.

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M. Thomsen, L. P . Rjder & A. Svejgaard

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Received 23 February 1976

22. Reports

from a Mixed Lymphocyte Culture Workshop. Tissue Antigens 4, 453, 1974. 23. Rood, J. J. van, Leeuwen, A. van, Keuning, J. J. & Blusst van Oud Alblas, A. The serological recognition of the human MLC determinants using a modified cytotoxicity technique. Tissue Antigens S, 73, 1975. 24.Ryder, L. P., Thomsen, M., Platz, P. & Svejgaard, A. Data reduction in LD-typing. Histocompatibility Testing, Munksgaard, Copenhagen, 1975, pp. 557-562. 25. Sachs, J. A., Sterioff, S., Robinette, M., Wolf, E., Curry, H. L. F. & Festenstein, H. Ankylosing spondylitis and the major histocompatibility system. Tissue Antigens 5, 120, 1975. 26. Scientific Tables. Ciba-Geigy 7th ed., 1970, p. 160. 27. Sheehy, M. J., Sondel, P. M., Bach, M. L., Wank, R. & Bach, F. H. HLA LD (lymphocyte defined) typing: a rapid assay with primed lymphocytes. Science, N.Y. 188, 1308, 1975. 28. Sondel, P. M., Sheehy, M. J., Benike, C., Alter, B. J. & Bach, F. H. Specific detection of HLA-D antigens using primed LD typing (PLT). Transplant. Proc., in press. 29. Svejgaard, A., Hauge, M., Jersild, C., PIatz, P., Ryder, L. P., Nielsen, L. Staub & Thomsen, M. The HLA system. An introductory survey. Monographs in Human Genetics 7, 1975. 30.Thomsen, M., Jakobsen, B., Platz, P., Ryder, L. P., Nielsen, L. Staub & Svejgaard, A. LDtyping, polymorphism of MLC determinants. Histocompatibility Testing, Munksgaard, Copenhagen, 1975, pp. 509-518. 31. Thomsen, M., Morling, N., Platz, P., Ryder, L. P., Nielsen, L. Staub & Svejgaard, A. Specific lack of responsiveness to certain HLA-D (MLC) determinants with notes on primed lymphocyte typing (PLT) . Transplant. Proc., in press. 32.Thomsen, M., Platz, P., Andersen, 0. Ortved, Christy, M., Lyngsere, J., Nerup, J., Rasmussen, K., Ryder, L. P., Nielsen, L. Staub & Svejgaard, A. MLC typing in juvenile diabetes mellitus and idiopathic Addison's disease. Tramplant. Rev. 22, 1 2 5 , 1975. 33. Thorsby, E. The human major histocompatibility system. Transplant. Rev. 18, 51, 1974. 34. Thorsby, E., Bondevik, H., Helgesen, A. & Hirschberg, H. Lymphocyte activating determinants of the human major histocompatibility system (MHS).Transplant. Proc. 7, 87, 1975. 35. Thorsby, E. & Solheim, B. Personal communication. 36. Tweel, J. G. van den, Bluss6 van Oud Alblas, A., Keuning, J. J., Goulmy, E., Termijtelen, A., Bach, M. L. & Rood, J. J. van. Typing for MLC(LD). I. Lymphocytes from cousin marriages offspring as typing cells. Transplant. Proc. 5 , 1535, 1973.