Tissue Antigens (1975), 5, 155-164 Published by Munksgaard, Copenhagen, Denmark

No part may be reproduced by any process without written permission from the author(s)

Histocompatibility Testing in Dogs. 11. Leukocyte Typing in Relation to the Mixed Lymphocyte Culture (MLC) Reactivity* S. F. GOLDMANN, K. KRUMBACHER, H. P. SCHNAPPAUF, R. P. HUGETAND H.-D. FLAD Department of Clinical Physiology, University of Ulm, UlndDonau, W. Germany The correlation between MLC reactivity (LD) and serological leukocyte typing (SD) was studied in a beagle colony. Disparity for a serologically defined non-DL-A lymphocyte antigen did not correlate with MLC reactivity. Lymphocytes of colony members with common ancestors and SD identical DL-A haplotypes did not stimulate each other i n the MLC. This implies that L D typing in the beagle colony can be generally predicted by DL-A SD typing. Consequently, lymphocytes of sibs homozygous for a given DL-A SD haplotype could be shown, with few exceptions, to be also homozygous for MLC determinants. Cells of these homozygous sibs can be used in MLC typing as reference cells for DL-A LD specificities. Two exceptions to the expected linkage between DL-A SD typing and MLC reactivity were found. These findings could not be explained by recombination within the DL-A region assuming a single major LD locus coding for MLC. Thus, suggestive evidence for more than one single LD locus has been obtained.

Received for publication 14 August 1973, accepted 13 January I975

The major histocompatibility region ( M HR) has been defined in several species. I n man, mouse and monkey the MHR (HL-A, H-2 and RhL-A respectively) includes serologically defined (SD) loci as well as lymphocyte defined ( L D ) loci (Kissmeyer-Nielsen et al. 1970, Dausset et al. 1971, Yunis & Amos 1971, Eijsvoogel et al. 1972, Dupont et al. 1973, Snell et al. 1971, Bach et al. 1973, Elkins & Bach 1973,

* Supported

Balner et al. 1973, Balner & Toth 1973). The LD determinants induce lymphocyte proliferation in the mixed lymphocyte culture (MLC), so far only preliminary evidence exists that LD specificities can be detected by serological means (van Leeuwen et al. 1973). The MHR of dogs seems to contain two closely linked SD loci, which were designated as the first and second DL-A SD

by Euratom, Fraunhofer Gesellschaft and Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 112 ) .

156

GOLDMANN ET AL.

locus, respectively (Vriesendorp et al. 1971, 1972, 1973).I t has been shown by Templeton & Thomas (1971) that MLC reactivity in dogs is governed by products of one chromosomal region. Van der Does et al. (1973) demonstrated that MLC reactivity in related dogs can be predicted by DL-A SD typing. Preliminary evidence for the existence of a separate LD locus (or loci) in the DL-A region has been provided in a few experiments (Vriesendorp et al. 1973, van der Tweel et al. 1975). The correlation between MLC reactivity and DL-A SD typing as well as a serologically defined non-DL-A antigen in a beagle colony is described in the current paper. Two MLC specificities could be defined. Possible evidence for more than one single LD locus is presented.

were incubated at 39' C together with an equal number of X-irradiated stimulating cells in 1 ml supplemented MEM-S medium containing 20 "/o pooled dog serum. Triplicate cultures of each cell combination were set up on at least two or three different occasions. Cultures were harvested after 144 h. Approximately 18-20 h before harvest 0.18 pCi thymidine-2-14C (Amersham, spec. act. 62 mCi/mMol) were added to each culture. The cultures were processed for scintillation counting by a NaOH digestion procedure. Values are given as the mean cpm (counts per minute) of three parallel cultures or as a stimulation ratio which is calculated from the cpm of the cell mixture in question divided by the cpm of the autologous control. A stimulation ratio < 2 was considered to be non-stimulatory.

Materials and Methods Tissue Typing Tissue typing was performed using the microdroplet lymphocyte cytotoxicity technique described by Mittal et al. (1968). The following DL-A antigens were determined employing sera tested at the first international workshop of canine immunogenetics (Vriesendorp et al. 1973) : DL-A 1, 2, 3, 7, 8, 9 and 10 of the first DL-A SD series and D G A 4 , 5, 6, 11, 12, 13 and 14 of the second DL-A SD series. I n addition, one serologically defined non-DL-A lymphocyte antigen (Ulm 55 = S 04003, Vriesendorp et al. 1973) was determined using the same technique.

Results The dogs investigated originate from a closely bred colony of beagles. The colony was started with four sires and 11 dames from the same kennel where they could be traced as third generation progeny of the original two sires and seven dames. The frequencies of the DL-A SD haplotypes found in 21 genotyped breeders of this colony are given in Table 1. High frequencies of the SD haplotypes 2,4 or 2,5 are apparent. The breeding schedule used in the colony resulted in numerous offsprings homozygous for the SD haplotypes DL-A

MLC Test The MLC method we have used in these studies is described in detail elsewhere (Goldmann & Flad 1975). I n brief, lymphocytes were separated from defibrinated blood using the Ficoll-Isopaque (spec. grav. 1080 mg/ml) gradient centrifugation (Boyum 1968). 1.5X 105 responding cells

DL-A haplotype

Total: 4 haplotypes

N

Frequency

42

0.999

157

LEUKOCYTE TYPING AND MLC REACTIVITY IN DOGS N.116

N- 105

N: 3 L

N: 116

20 -

15

-

13.9 13.:

...

11.L 10-

...... ... ...

5-

"

11

........... *.::::La

....... .......... ..:iB:.. .... ........... ........ ........... .... ...

........... .......

________~~~~__

.......... .... ::;gZl< ......... .... ...... :::... :::.

1.31

1 DL-A JDENTICAL

ONE HAPLOTYPE

DIFFERENCE

SIBS OR RELATED "

-

COMPATIBLE

(ala

"

a/ b )

ONE HAPLOTYPE DIFFERENCE (sib- a / a 1 SIBS OF?

TW -O HAPLOTYPE DIFFERENCE SIBS

TWO HAPLOTYPE

-~ DIFFERENCE UNRELATED

RELATED

5185 O R . RELATED

Figure 1 . MLC stimulation ratios in DL-A SD typed dog pairs.

2,4 or 2,5 as well as heterozygous for both haplotypes. In order to determine the relation between the DL-A SD antigens or haplotypes and the reactivity in the MLC, mixed lymphocyte cultures were performed using

cell combinations of colony as well as unrelated beagles. T h e overall results are shown in Fig. 1 where MLC combinations between cells of non-sibs but colony members (half-sibs, parent-child, cousin combinations, etc. ) are designated as related.

158

GOLDMANN ET AL.

Table 2 M L C rpactivity in relation to a serologically dejned non-DL-A antigen disparity a, b Stimulating cells

I

-

I Non-DL-A I

Ax

I

7,-

V

z

I

55 -

B

U

2,5

I

PHA

13203k564 14274f1904 (36.3)

7,-

Fr:

Ux

12816*1057 17127k213 (14.5)

(0.3)

2,4

B

3 c

Bx

2,4

A

(li

3

D1,-A

55+

9940 i.2757 7912 i; 1129 (59.2) (47.1)

3,-

168 f 41 (1.0)

18583i1613

standard deviation and stimulation ratios in parentheses. a cpm b U non sib, colony beagle. 1

Table 3 MLC-stimulation in cell combinations of S D heterozygous and homozygous sibs a, Stimulating cells

I SA SB

3 (li

DL-A 2,4

V

M

sc

.f

-3

SD U

I

SBx

1

SCX

1

SDx

I

Ux

725 f 53 (1 .O)

553 f 102 4812 i.362 (0.8) (6.6)

4763 i 411 (6.6)

9394 i 651 (13.0)

2,4

598 i 124 (1.1)

527 f 158 (1 .O)

4219 f 731 (8.0)

9747 ir 149 (18.4)

9790 i- 147 (18.6)

2,5

3161 -+ 479 (4.9)

3096 f 594 (4.8)

645 t 87 (1 .O)

3208 i:852 (5.0)

5041 i 472 (7.8)

2,5

563 i- 97 (1.9)

427 C 102 (1.5)

401 t 39 (1.4)

291 2 70 (1.O)

4848 i:423 (16.7)

7,-

4260 i 148 (12.8)

531 JI 73 (1.6)

3561 i 320 (10.8)

332 i- 84 (1.O)

2,5

8

B Fr:

SAX

2,4 2,4

e

I

2,4 2,5

4833 f 74 (14.4)

a cpm i:standard deviation and stimulation ratios in parentheses. b

U

=

non sib, colony beagle.

Cells of beagles with DL-A SD haplotype disparities in both directions stimulated in all cell mixtures tested. Cells from DL-A identical sibs or related beagles with phenotypic identical SD haplotypes did not stimulate each other in 105 combinations (Fig. 1) and could therefore be considered genotypically SD and LD identical. A detailed example of such a combination is given in Table 2. Cells of DL-A SD homozygous beagles (a/.) did not stimulate

in general cells of their heterozygous sibs which shared one haplotype and differed in the other or cells of related heterozygous colony beagles (a/b, a/c, etc.). These homozygous cells were stimulated by cells of the heterozygous dogs in over 100 MLC combinations performed (Fig. 1) . The exceptions concerning cells of two colony beagles will be described later. SD homozygous beagles could, therefore, with two exceptions, be considered homozygous for

159

LEUKOCYTE TYPING A N D MLC REACTIVITY IN DOGS

Table 4 MLC-stininlation ratios in family 1 a, 0, C i d

DL-A

-8

SA, SC, SD, SE, M

295

SB

2, s

v)

(SA, SC, SD,SE, M) x

SBX

c"

3

k

SG,SH

2

F U

ux

6.22 (4.1-9.1)

16.66 (6.2-33.6)

0.83 (0.6-1.3)

0.95 (0.8-1.1)

1.o

2,5

1.6

2.2 1.o

2,s

1.75 (1.5-1.9) 1.57 (0.6-1.9)

1.5 (1.0-1.9)

1.15 (0.7-1.6)

1.o

1.9

2,s

13.5 (12.9-14.1)

1.56 (1.1-1.8)

1.8

0.8

0.93 (0.7-1.3)

1.o

10.9

2,s

6.78 (4.0-8.9)

9.2

6.6

4.8 (4.7-4.9)

8.1

1.o

3,-

2,4 2,4

2,4

8.76 8.14 (4.7-15.4) (3.5-17.6)

Fx

1.46 (0.5-1.9)

2,4

@

(SG, SH)x

2,s

3

SF

SFx

PI (1.3-1.9)

1.75 (1.1-1.4)

7.6 2'9 1.9

20.1

-

a Siblings A-H = SA-SH b Sire = F C Dame = M d U = non sib, colony beagle

the whole DL-A region, hence the suggest- cause MLC stimulation. An example is ed LD gene (or genes). The results of such given in Table 2. In two families, exceptions to the DL-A an experiment are presented in Table 3 and demonstrate that sibs SA and SB ho- SDJMLC linkage were found. No stimulamozygous for the DL-A SD haplotype 2,4 tion between cells of sibs, despite SD disare also homozygous for a closely linked parity, was found in one family (Fig. 1, MLC gene (or genes) preliminarily desig- Table 4 ) . I n a second family, cells of a SD nated as DL-A LD 1. The correspond- homozygous beagle (a/a) stimulated the ing MLC gene (or genes) of the homo- cells of his haploidentical heterozygous sibs zygous beagle SC (DL-A SD 2,5/2,5) was (a,b, Fig. 1, Table 5 ) . The phenotype of the common sire ( F in preliminarily designated as DL-A LD 11. Cells of these homozygous sibs can be used Tables 4 and 5 ) of both families was found in MLC typing as reference cells of DL-A to be DL-A SD 2,4,5. Crossing this sire LD specificities. Beagles homozygous for with a dame carrying the antigens DL-A the SD haplotypes 3,- or 7,- were not SD 2,3,4 revealed offsprings with the genotypes DL-A SD 2,413,- and 2,5/3,- in found in our colony. I n all combinations tested, a serological- three out of 11 pups each. The genotype ly defined non-DL-A disparity determined of the sire can therefore be deduced to be by lymphocyte antiserum Ulm 55 did not DL-A SD 2,4/2,5.

160

GOLDMANN ET AL.

Table 5 MLC-stimulation ratios in family Zag by C , d

SA,SB,SC, SD,SE,SF

-

SB’

DLA

scJSD’ SE, SF)x

2,4

1.3 (0.5-1.9)

2,5

(SG, SH, SK, M)x

SIX

Fx

ux

5.97

X

SG,SH,SK, 2,4 M 2,4

20.99 (4.141.6)

1.21 (0.8-1.8)

1.43 (1 .O-1.8)

8

SI

11.35 (4.6-19.1)

0.85 (0.7-1.0)

1 .o

14.8

33.4

2,4

1.35 (0.9-1.5)

1.2 (1.1-1.3)

1.1

1 .o

8.6

2,s

12.3 (5.9-19.6)

11.1 (9.0-15.0)

20.5

11.7

1 .o

7,-

yl

3

2,4

0

E F p:

2,4

U

2,s

a Siblings A-K

=

12.85 15.8 (12.4-17.4) (11.1-20.6)

SA-SK

Sire = F Dame = M U = non sib, colony beagle

The mother ( M ) in family 1 is due to phenotyping DL-A SD 2,5. Of the four possible haplotypes (2,5; 2,-; -,-; , 5 ) the haplotypes 2,- and -,- are very unlikely since by crossing this dame with a DL-A SD 2,4/2,5 sire 0/19 pups had the phenotype 2,4. The haplotype -,5 cannot be formally excluded. I t has, however, never been found in other genotyped beagle colonies so far, whereas 2,5 is very common. Thus, the most probable genotype of the mother is homozygous (DL-A SD 2,5/2,5). Sibs (S) of family 1 (Table 4 ) are consequently SD homozygous DL-A 2,5/2,5) or heterozygous (DL-A 2,4/2,5). Table 4 demonstrates the MLC results obtained in family 1. As expected, cells of the homozygous dame ( M ) or sibs SA-SE (DL-A SD 2,5/2,5) did not stimulate the cells of the heterozygous family members F, SF-SH (DL-A SD 2,4/2,5) while they could be stimulated by cells of these he-

terozygous family members. Exceptions were observed where the cells of the homozygous sib SB (DL-A SD 2,5/2,5) could not be stimulated by the cells of the heterozygous sibs SG and SH (DL-A SD 2,4/ 2,5). I n addition, only a borderline MLC response of cells of this homozygous sib SB occurred in the MLC combinations with the cells of the heterozygous sib SF and the heterozygous sire F (both DL-A SD 2,4J 2,5). These data suggest that sib SB did not inherit the same LD genes as his SD identical sibs SA, SC, SD and SE or that sibs SG and SH may not be MLC identical with the SD identical sib SF. Under this assumption, one could expect that stimulation occurred between cells of the SD identical sibs or in combination with their SD identical parent but this was not the case (Table 4 ) . The MLC results obtained demonstrated in Table 4 were found to be reproducible in subsequent repeats even

LEUKOCYTE TYPING AND MLC REACTIVITY I N DOGS

when different numbers of stimulating cells were used. The phenotype of the dame ( M ) in family 2 (Table 5) was found to be DL-A SD 2,4. The dame was considered to be genotypical homozygous (DL-A SD 2,4/ 2,4) although the genotype 2,4/-,4 alone could not be formally excluded by segregation analysis. Sibs (S) of family 2 (Table 5) are consequently SD homozygous (DLA SD 2,4/2,4) or heterozygous (DL-A 2,4J 2,5). As expected cells of the homozygous family members M, SG, SH and SK did not stimulate the cells of their heterozygous sibs SA-SF and the sire F and coud be stimulated by cells of these heterozygous family members (Table 5). Unexpected MLC results were found where cells of the homozygous sib SI (DL-A SD 2,4/2,4) stimulated the cells of the haploidentical heterozygous sibs SA-SF (DL-A SD 2,4/ 2,5). These data suggest that SI did not inherit the same LD genes as his SD identical sibs SG, SH and SK or that SI is not a family member at all. Under this assumption one could expect that stimulation occurred between the cells of SI and cells of the SD identical beagles, but this was not observed (Table 5). Subsequent repeats showed substantially the same pattern of MLC reactivity as described in Table 5 even when different numbers of stimulating cells were used. Discussion Preliminary evidence for a separate lymphocyte defined MLC locus (or loci) linked to the DL-A SD loci has been provided in a few experiments (Vriesendorp et al. 1973, van der Tweel et al. 1975). As our study and that of van der Does et al. ( 1973) indicate, dog lymphocyte proliferation in MLC appears, in general, to follow the genetic pattern of human and mouse

161

MLC (Yunis & Amos 1971, Bach et al. 1973), since cells of SD identical sibs usually do not stimulate each other. A particular feature of the closely-bred colony at our disposal concerns the situation in which certain families possess only two genetically different haplotypes. Consequently, several family members are homozygous or heterozygous for the given haplotypes. As described in our study, cells of sibs homozygous for the DL-A SD haplotypes 2,4 or 2,5 in general did not stimulate cells of their heterozygous sibs in the MLC although they could respond to cells of these heterozygous sibs. The SD homozygous sibs could, therefore, be considered homozygous for the LD gene (or genes) of the DL-A region. This enables us to use their cells as a reference panel for MLC typing in dogs as described recently by Grosse-Wilde et al. (1975). Attempts to compare MLC specificities were discussed at the second international workshop of canine immunogenetics (Goldmann 1975, personal communication) and will be carried out in the near future. In two beagle families exceptions to the DL-A SD/MLC linkage were confirmed by subsequent experiments (Tables 4 and 5). The following interpretations of these data will be discussed :

I . Pre-Immunization Interference of blocking antibodies or presensitized lymphocytes in the MLC is unlikely since sibs and the sire of both families were not immunized by transfusions, pregnancies, experimental immunization trials or transplantation procedures (Eijsvoogel 1971, Eijsvoogel et al. 1971, Hattler et al. 1972, Miller et al. 1971, 1972, 1973a). I I . Multiple DL-A L D Loci The reciprocal MLC non-reactivity observed between cells of the SD identical sibs seems to indicate that SD similarity

162

GOLDMANN ET AL.

within both families comprises, as expected, LD identity. Differences between the DL-A SD identical homozygous sibs are revealed, however, when their cells are cultured together with cells of heterozygous sibs. These findings cannot be explained on the basis of a single major DLA LD locus coding for MLC. However, they could be compatible with the hypothesis that the DL-A region comprises two or more LD loci. Two or even multiple LD genes within the M H R were suggested recently in man and mouse (Eijsvoogel et al. 1972, Dupont et al. 1973, 1974, Thorsby et al. 1973, Meo et al. 1973). The low stimulation ratios observed in cell combinations under discussion are comparable to the weak MLC reactivity described recently in similar cell mixtures in man (Eijsvoogel et al. 1972, Sasportes et al. 1973). I t was suggested that this pattern of MLC reactivity represents a second (or more) weak LD locus (or loci) in the M H R (Dupont et al. 1973, 1974). Whether each gene independently is coding for weak MLC determinants or whether multiple genes contribute to MLC in cumulative fashion is unknown at present. Both mechanisms are compatible with the model of multiple DL-A LD loci and could explain the unexpected MLC results.

Existence of a “Weak” Non-DL-A Region The existence of a LD region not linked to the MHR is well established in the mouse (Festenstein & Demant 1973).An epistatic effect of genes not linked to DL-A seems to be ruled out in our colony since stimulation in cell mixtures of DL-A identical sibs was not observed. The unexpected results, however, were obtained in cell combinations of SD genotypically different sibs. Whether or not this is due to the influence of “weaker” MLC loci outside or inside DL-A remains to be assessed.

Recessive M L C Genes The MLC results in family 2 are in agreement with the hypothesis that recessive L D genes will contribute to MLC only in a situation of homozygosity where they are in double dose. Known instances of recessivity, however, are rather few in immunogenetics and only further genetic characterization of LD gene products can provide a conclusive answer as to whether or not recessive L D genes actually exist (Lebrun et al. 1973). Cross-Reactivity of L D Genes Cross-reactivity and antigen inclusion are well established phenomena in SD immunogenecity (Svejgaard & Kissmeyer-Nielsen 1968, Kissmeyer-Nielsen & Thorsby 1970). Both phenomena were recently described between LD determinants in man (Dupont et al. 1973, Keuning et al. 1975). This could explain the borderline or negative MLC response of SB in family 1 but it would seem to be more prudent to wait for further evidence before establishing crossreactivity of the L D determinant in dogs. Further MLC experiments in beagles and a planned breeding of sibs and families in question will have to be performed before the exact role of the suggested explanations can be reexamined. Acknowledgements We are grateful to Dr. H. M. Vriesendorp, Rijswijk, for kindly providing DL-A antisera. We wish to thank Mrs. J. Flad, Mrs. A. Geist, Mrs. A von Neubeck and Miss F. Schneider for their excellent technical assistance.

References Bach, M. L., Widmer, M. B., Bach, F. H. & Klein, J. (1973) Mixed lymphocyte cultures and immune response region disparity. Transplant. Proc. 5, 369-375.

LEUKOCYTE TYPING AND MLC REACTIVITY IN DOGS

Balner, H., Gabb, B. W., Toth, E. K., Dersjant, H. & van Vreeswijk, W. (1973) The histocompatibility complex of rhesus monkeys. I. Serology and genetics of the RhL-A system. Tissue Antigens 3, 257-272. Balner, H. & Toth, E. K. (1973) The histocompatibility complex of rhesus monkeys. 11. A major locus controlling reactivity in mixed lymphocyte cultures. Tissue Antigens 3, 273290. Bias, W. B., Hopkins, K. A., Hutchinson, J. R. & Hsu, S. H. (1974) Evidence for an unlinked gene which modifies HL-A antigen expression. Tissue Antigens 4, 36-41. Beyum, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, Suppl. 97 Dausset, J. (1971) The genetics of transplantation antigens. Transplant. Proc. 3, 814. Dausset, J., Rapaport, F. T., Cannon, F. D. & Ferebee, J. W. (1971) Histocompatibility studies in a closely-bred colony of dogs. 111. Genetic definition of the DL-A system of canine histocompatibility, with special reference to the comparative immunogenecity of the major transplantable organs. J. exp. M e d . 134, 1222-1237. van der Does, J. A., van Rood, J. J., Walker, W. S. & Epstein, R. B. (1973) Consequent intrafamilial immunization for DL-A haplotyping in canines. J. exp. M e d . 137, 494-503. Dupont, B., Jersild, C., Hansen, G. S., Staub Nielsen, L., Thomsen, M. & Svejgaard, A. (1973) Multiple MLC ( L D ) determinants on the same HL-A haplotype. Transplant. Proc. 5, 1481-1487. Dupont, B., Good, R. A., Hansen, G. S., Jersild, C., Staub Nielsen, L., Park, B. H., Svejgaard, A., Thomsen, M. & Yunis, E. J. (1974) Two separate genes controlling stimulation in mixed lymphocyte reaction in man. Proc. nut. Acad. Sci. ( W a s h . ) 71, 52-56. Eijsvoogel, V. P., Schellekens, P. Th. A., BreurVriesendorp, B., Koning, L., Koch, C., van Leeuwen, A. & van Rood, J. J. ( 1971) Mixed lymphocyte cultures and HL-A. Transplant. Proc. 3, 85-88. Eijsvoogel, V. P. (1971) Lymphocyte stimulation and inhibition in vitro. Studies on HL-A and an anti-lymphocyte serum (ALS) . Thesis, Amsterdam. Eijsvoogel, V. P., van Rood, J. J., du Toit, E. D. & Schellekens, P. Th. A. (1972) Position of a locus determining mixed lymphocyte reaction distinct from the known HL-A loci. Eur. J. Immunol. 2, 413418.

163

Elkins, W. L. & Bach, F. H. (1973) Danger of doctrinal tyranny in the terminology of H-2. Cell. I m m u n o l . 7, 522-524. Epstein, R. B., Storb, R., Ragde, H. & Thomas, E. D. (1968) Cytotoxic typing antisera for marrow grafting in littermate dogs. Transplantation 6, 45-58. Festenstein, H. & Demant, P. (1973) Workshop summary on genetic determinants of cell-mediated immune reactions in the mouse. Transplant. Proc. 5, 1321-1327. Goldmann, S. F. & Flad, H.-D. (1975) Histocompatibility testing in dogs. I. A semi-micro mixed lymphocyte culture (MLC) technique for histocompatibility matching in dogs. Tissue Antigens 5, this issue. Grosse-Wilde, H., Vriesendorp, H. M., Netzel, B., Mempel, W., Kolb, H. J., Albert, E. D. & Westbroek, D. L. (1975) Immunogenetics of 7 LD alleles of the DL-A complex in mongrels, beagles and labradors. Transplant. Proc., (in press). Hattler, B. G., Miller, J. & Johnson, M. C. ( 1972) Cellular and humoral factors governing canine mixed lymphocyte cultures after renal transplantation. 11. Cellular. Transplantation 14, 47-56. Keuning, J. J., Termijtelen, A., van der Tweel, J. G. & van Rood, J. J. (1975) Typing for LD (MLC). Transplant. Proc., (in press). Kissmeyer-Nielsen, F., Staub Nielsen, L., Lindholm, A., Sandberg, L., Svejgaard, A. & Thorsby, E. (1970) The HL-A system in relation to human transplantation. Histocompatibility Testing 1970, p. 105-135. Munksgaard, Copenhagen. Kissmeyer-Nielsen, F. & Thorsby, E. ( 1970) Human transplantation antigens. Transplant. R e v . 4, 44-49. Lebrun, A., Sasporters, M. & Dausset, J. (1973) Role of MLC locus and related genes of chromosomal HL-A region. Transplant. Proc. 5, 363-367. van Leeuwen, A., Schuit, H. R. E. & van Rood, J. J. (1973) Typing for MLC ( L D ) : 11. The selection of nonstimulator cells by MLC inhibition tests using SD-identical stimulator cells (MISIS) and fluorescence antibody studies. Transplant. Proc. 5, 1539-1542. Meo, T., Vives, J., Miggiano, V. & Schreffler, D. (1973) A major role for the Ir-1 region of the mouse H-2 complex in the mixed leucocyte reaction. Transplant. Proc. 5, 377-381. Miller, J., Hattler, B., Davies, M. & Johnson, M. C. (1971) Cellular and humoral factors governing canine mixed lymphocyte cultures

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after renal transplantation I. Antibody. Transplantation 12, 65-75. Miller, J., Hattler, B. G. & Johnson, M. C. (1972) Cellular and humoral factors governing canine mixed lymphocyte cultures after renal transplantation. 111. Further studies with antibody. Transplantation 14, 57-64. Miller, J., Rood, F., Lifton, J. & Hattler, B. G. (1973a) Cellular and humoral factors governing canine mixed lymphocyte cultures after renal transplantation IV. Second-set transplants and cytophilic antibody. Transplantation 16, 142-150. Miller, J., Howard, R. J., Hattler, B. G. & Najarian, J. S. (1973b) Correlation of MLC reactivity after experimental and clinical transplantation. Virus and cytophilic antibody: sources of false negatives. Transplant. Proc. 5, 1771-1774. Mittal, K. K., Mickey, M. R., Singal, D. P. & Terasaki, P. 1. (1968) Serotyping for homotransplantation. XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6, 913-927. Sasportes, M., Mawas, C., Bernard, A., Christen, Y. & Dausset, J. (1973) MLR in families with an HL-A haplotype shared by parents: recombination between SD 2 and LD 2 and possible evidence for an LD 3 locus. Transplant. Proc. 5, 1517-1522. Snell, G. D., Cherry, M. & Demant, P. (1971) Evidence that H-2 private specificities can be arranged in two mutually exclusive systems possibly homologous with two subsystems of HL-A. Transplant. Proc. 3, 183-186. Svejgaard, A. & Kissmeyer-Nielsen, F. (1968) Cross-reactive human HL-A isoantibodies. Nature (Lond.) 219, 868-869. Templeton, J. W. & Thomas, E. D. (1971) Evidence for a major histocompatibility locus in the dog. Transplantation 11, 429-431. Thorsby, E., Hirschberg, H. & Helgesen, A. (1973) A second locus determining human

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Dr. S. F. Goldmann Department of Clinical Physiology University of Ulm D-7900 Ulm/Donau Steinhoevelstrasse 9 W. Germany