Tissue Antigens (1974), 4,495-506

Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

Mixed Lymphocyte Culture Technique Standardization of a Test-System with lo5 Responding and

10’ Stimulating Lymphocytes per 1 ml

MOCENS THOMSEN,~ GRETE S. HANS EN,^ ARNE SVEJCAARD,~ CASPER J E R S I L D , ~ ? ~ JOHN A. HANS EN,^ R. A. GOOD^ AND Bo D U P O N T V

‘Tissue Typing Laboratory, Blood Bank and Blood Grouping Department of the State University Hospital (Rigshospitalet), Copenhagen, Denmark; and

Clinical Immunology Laboratory and Tissue Typing Laboratory, Memorial Sloan-Kettering Cancer Center, New York, U.S.A.

An MLC test system is described utilizing 102 responding and 100 stimulating lymphocytes in a total of 1 ml of culture medium. Some of the variables of the test have been evaluated and the reproducibility of the test within a given day and between days investigated ac- cording to a Workshop protocol. This system was first applied to three groups: HL-A identical siblings, one haplotype different family members and two haplotype different individuals. Within each group, the log converted stimulation ratios are normally distributed, allowing the use of simple statistical analysis in repeated testings. In addition, a group of weakly positive MLC reactions can be detected. This group includes the LD identical but SD different sibling combinations. Within the same group we find some unrelated HL-A sero-identical combinations and the so-called LD typing response, used to identify LD deter- minants by means of LD and SD homozygous stimulating test cells.

I t is concluded that this MLC test system allows identification of identical combinations, weakly positive combinations and one and two haplotype responses even within unrelated combinations. This test system may have advantages over the microtiter test system because the large culture volume gives a low concentration of blastogenic factors in typing responses.

Received for publication 24 May, accepted 24 June 1974

Supported in part by grant 512-2507 from the Danish State Research Council. Part of the work was performed during Bo Dupont’s appointment as research fellow at Department of Pediatrics and Pathology, University of Minnesota Hospitals, Minneapolis, Minnesota, U.S.A.

496 REPORTS FROM AN M I X WORKSHOP

As recently reviewed by Dupont et al. ( 1974), studies of families with recombina- tions within the major histocompatibility system (MHS) have made it possible to map this chromosomal region. The stimu- lation in the mixed lymphocyte culture (MLC) is controlled by genes of at least two separate genetic loci: one strong one located outside the HL-A region close to the FOUR locus and a weaker one possib- ly located between the LA and FOUR loci. The products of these lymphocyte defined (LD) genes are as yet not demonstrable by serological means, and in order to cha- racterize them further, it has become ne- cessary to develop reliable micro-test sy- stems for large scale application of MLC tests. The test system used should allow discrimination between negative and posi- tive reactions and preferably also make it possible to analyse quantitatively the de- gree of positivity in the reactions.

It is well established that the majority of HL-A identical sibling combinations do not provoke any stimulation in MLC. Analysis of MLC data obtained in siblings, identical for the strong LD locus, but dif- fering by serologicaUy detectable (SD) antigens has revealed a weak positive sti- mulation (Dupont et al. 1974). Such weak MLC reactivity is also seen in approxi- mately 10 % of HL-A identical unrelated MLC combinations (van Rood & Eijs- voogel 1970, Ssrensen & Nielsen 1970, Mempel et al. 1973). Already in 1967, Bach & Amos had shown that the MLC reaction could be used as a measure of the degree of histocompatibility, because two haplotype different sibling combi- nations on an average stimulated twice as much as one haplotype different combina- tions. Ideally, this approach should be ex- panded to also include discrimination be- tween one and two haplotype differences in unrelated individuals.

In this report, we describe the method

we have applied to MLC testing during the past three years, with minor modifica- tions. It is a micro-MLC test using 105 responding and 105 stimulating lympho- cytes in 1 ml of culture volume. The sys- tem has made it possible to satisfy most of the above mentioned demands to an MLC test system.

Materials and Methods For the lymphocyte cultures and isolation proce- dures RPMI 1640 (Biocult, Glasgow) is used, containing:

bicarbonate 0.85 g/l Hepes buffer 20 mn? streptomycin 0.5 mg/ml penicillin 500 IU/ml heparin (NOVO, without preservation)

glutamin 1.5 uM

This mixture will henceforth be referred to as medium. As serum source, we use a pool ob- tained from 10-20 healthy non-transfused male donors. The lipidaemic sera are excluded and none of the pool contained cytotoxic antibodies. The serum pool is distributed into sterile WR plastic tubes (Nunc, Roskilde, Denmark) and stored at -20" C until use. Blood-drawing, transport of blood. Peripheral blood is drawn in a closed system aseptically into a sterile bottle containing an equal amount of medium. The blood and medium is carefully mixed by gently shaking the bottle.

The transportation of blood-medium mixture takes place a t room temperature and in this way the majority of lymphocytes are kept viable for at least 24 h, allowing shipment of fresh samples over long distances. When blood samples arrive in the afternoon the lymphocytes are usually isolated the same day and resuspended in me- dium with 15 % serum. They are then kept at 37" C until they are used in MLC the next day. Isolation of lymphocytes. Six ml blood-medium mixture is layered on 3 ml lymphoprep (Nye- gird, Oslo) in plastic WR tubes. The tubes are capped and spun for 30 min with approximately 600 g a t the interface. The lymphocyte-rich layer is harvested with a Pasteur-pipette, and the cells are washed three times ( 5 min, 600 g) with medium containing 5 5% serum and finally resuspended in medium containing 15 % serum.

15 IUJml

REPORTS FROM AN M1.C WORKSHOP 497

Countzng of lymphocytes. Twenty-five p1 of each suspension are stained and the lymphocytes are counted in a Neubauer counting chamber, ex- cluding morphologically damaged cells and gra- nulocytes. The percentage of granulocytes is usu- ally less than 1 % and the viability of the lym- phocytes (Trypan blue exclusion test) above 95 %. The contamination with monocytes is usually 5-10 % as assayed by incubation with latex par- ticles. The recovery from fresh samples is usually above 95 9'0 of the mononuclear cells, whereas the recovery can drop to about 50 %, when 24-h-old samples are isolated. The viability, how- ever, is always above 95 %, indicating that the dead cells are eliminated during the isolation procedure.

Preparation of stimulating cells. During the major part of the study the lymphocytes used as stimulating cells were treated with mitomycin-C (Sigma) at a concentration of 40 pgimg at 37" C for 30 min. The cells are washed three times in medium with 5 % serum, resuspended and counted. An alternative way of producing stimulating cells is irradiation. During the last five months, we have used irradiation from a ' W o source (Atomic Energy Commission, Rim, Denmark). The dose-rate is 5,000 rad/min, and the cells are given approximately 2,000 rad.

Preparation of lymphocyte cultures, The con- centration of responding lymphocytes is 105 per 0.5 in1 of medium with 15 % serum. The sti- mulating lymphocytes are usually prepared in both a concentration of 105 and 3 x 100 cells per 0.5 ml. 0.5 ml of the responder cell sus- pension and 0.5 ml of the stimulator cell sus- pension are distributed into Nunc plastic tubes 10 X 37 mm, using a 500 pl Eppendorph pipette. All combinations are made in triplicates, the autologous combinations in tri- or sexplicate. T o ensure sufficient treatment of the stimulating cells, these are also cultured in all combinations; in no case did we observe stimulation in these combinations.

Culturing and labelling of the cultures. The tubes are placed in a water-jacketed incubator (Forma Scientific) with 5 9'0 C 0 2 in humidified air a t 37" C. Twenty-four h before termination of the cultures, we add 100 p1 aqueous solution of 2-*4C-thymidine, 0.1 ,uCi per tube, spec. act. 50 mCi/mmole (NEN Chemicals).

Termination of the cultures, harvesting and scintillation counting. The MLC test is usually terminated after 5 days, PHA cultures after 3 or

4 days. The cultures are placed at + 4" C, and harvested when convenient. The harvesting has been made either manually on Whatman GF/C glass fibre filters or in a multiple cell culture harvester (Skatron, Norway) with Gelman glass fibre filters. The cells are transferred to the fil- ter and washed with approximately 20 ml di- stilled water. After air drying, the filters are placed into plastic counting vials (Scintec, Gen- tofte, Denmark) and 5 ml Instagel (Packard) is added. The activity is counted in a Beckman liquid scintillation counter to an accuracy of 5 9' or maximally 1 min. The results are calcu- lated as mean cpm (counts per minute) of the triplicates minus background. Single cultures without activity (i.e. with cpm at the back- ground level) are excluded.

Expression of results. The results are usually expressed as stimulation ratios (SR=AB,/AA,), i.e. the cpm in the actual combination (AB,) divided by the cpm autologous combination ( AA,,,) . I n some cases we have used increments, i.e. actual cpm minus autologous cpm (AB,- AA,) . Another expression is the relative respon- se (RR) , i.e. the increment of the actual combi- nation divided by the increment in a supposedly two haplotype response (AU,). When two or more such combinations were included, the aver- age of the AU, combinations was used. (For further explanation see Joint report, Thorsby et al. this issue.)

Freezing procedure. The method used is a mo- dification of the method described by Wood et al. ( 1972). The essential points are described in the Joint Report (Thorsby et al. this issue) and will not be discussed here, as the technique has been changed in order to improve the recovery of lymphocytes.

Results Influence of amount of 14C thymidine added to the culture. Fig. 1 shows the cpm obtained in one MLC combination, when different dilutions of the thymidine are added. The volume was kept constant, and the spec. act. is constant (50 mCi/mM) . Each point represents the mean of six cul- tures, and the standard error of the mean is also indicated.

Influence of uarious irradiation doses. An LD-7a homozygous cell was used as sti-

498

10

REPORTS FROM AN M1.C WORKSHOP

h corn x lo*

-

-/! 0

“%”.I c o w

1 -/!

- ./: f- P

2

1 u C i Thymidine

Figure 2. The influence of the radiation dose on the stimulating and re- sponding capacity of different MLC combinations. A cell homozygous for HL-A 3,7 and LD-7a (Ax or Am) is used as stimulator. An LD-7a hetero- zygous cell (B) and a LD 7a negative cell (C) are used as responder cells. The irradiation doses of the cell Ax are indicated on the abscissa; and at the far right are shown the results in the same combinations when mito- mycin treatment is used instead (Am). The combination ABm gave the same day a cpm of 7,000, the PHA response of untreated A cells was 30,000 cpm (not shown on the Fig.).

For cultures stimulated with PHA-P (Difco) 105 cells in 0.5 ml volume are used, harvested on day 4. PHA concentration 20 pg per 0.5 ml. Label- ling for 24 h with 0.05 pCi 1% thymidine.

REPORTS FROM AN MLC WORKSHOP 499

mulator against an LD-7a heterozygous unrelated cell and against an LD-7a nega- tive cell. Different irradiation doses were used and mitomycin treated cells were in- cluded in the experiment. The results are shown in Fig. 2. The homozygous stimu- lator is named A, the heterozygous cell B and the LD-7a-negative cell C.

In the same experiment the combination ABm gave a mean cpm of 7,000 (not shown in Fig. 2 ) . I t appears that the re- sponse of the stimulating cell Ax towards the B cell is effectively blocked already with a irradiation dose of 500 rads and even irradiation doses of 5,000 rads give satisfactory stimulation (CAx) . I t also appears that the PHA induced prolifera- tive response is not totally eliminated even with high doses of irradiation, whereas the mitomycin treatment seems more effective in this respect.

The PHA response of the untreated A cells was 30,000 cpm.

Reproducibi l i ty of t h e T e s t Sys t em W o r k s h o p protocol la. Two individuals were bled and the blood samples divided as in Fig. 3. The Workshop protocol has been extended to include the comparison of results obtained by two different tech- nicians. Table 1 shows the statistical varia- tion in a nested analysis of variance. There

is significant difference between the com- binations but no significant difference be- tween technicians or within responding or stimulating cells.

Figure 3. The distribution of blood samples from persons A and B in the MLC split experiment (Workshop protocol part Ia).

The same procedure was done independently by two technicians (Ti and T2).

W o r k s h o p protocol I b included testing of the same MLC combinations on different days. We have repeated one experiment with four cell donors on four different days using new samples every time (Table 2a). Some of these combinations were known to be weakly positive. Table 2b shows the variation when the results are

Table 1 Log transformed CPM

Source of variation I Mean. var. 1 df I P I Variance components yo

Between combinations 1.646 1 0.0s 86 Between technicians 0.117 2 n.s. 3 Between responder cells 0.102 4 n.s. 4 Between stimulator cells 0.068 8 n s . 2 Within triplicates 0.128 32 n.s. 5

The statistical variance of one split experiment, evaluated by ANOVA (kindly performed by L. U. Lamm). It can be seen that the variance between responding cells, stimulating cells and technicians is smaller than within the triplicates.

500

Exper- iment

IIEI’ORTS FROM AN MLC WORKSHOP

l m 2m

b Responder and

HL-A

3m 1 4m

1: 3,7/1O,W18

2: 3,7/1O,W16

3: 2,10,5,MK

4: 3,7/3,7

I I1 111 I V I I1 111 IV I I1 111 IV I I1 I11 IV

(1105) (495) (548) (329) 588

1476 1041 389

8499 15300 13203 5975 1865 1480 3969 1662

(958) (947) (431) (488) 1413 2590 1678 879

14567 18911 14590 9286 3696 3122 4609 2322

6150 4660 4621 2389 (406) (663) (556) (170) 8979

10530 7301 4676 4580 5969 4348 2577

8361 8231 4354 2314 (356)

(1255) (341) (100)

16573 14204 8313 6773 8004 5147 6994 4586

8813 5349

13111 5487 7442 8009 6850 1900 (719)

(1 608) (1387)

(498) 6701 8167 9477 3058

15563 13056 12993 5377

11099 9959 9945 2039

(593) (1415) (1909)

(554) 9420

10258 18243 9939

1576 1500 2095 350 982

1241 1136 194

7360 1 SO96 9862 4283

(369) (318) (797) (192)

2068 2466 1935 429 973

2262 2064 433

11348 15553 10642

5042 308 242

1009 194

~

Results (cpm) of four different (I-IV) MLC tests between four different individuals ( 1 4 ) using 10: (a) and 3 x lo5 (b) stimulating cells.

Table 2 b Variation in repeated testings

I I lo5 stimulating cells mean coeff. of var.

3 x lo5 stimulating cells mean coeff. of var.

~ ~

range CPm 45 (32-56) SR 31 (13-57) log SR 25 (6-91) increment 54 (33-87) RR 53 (27-89)

range 41 (26-61) 48 (29-74) 30 (1 5-97) 43 (22-7 3) 53 (18-103)

The variance of repeated ML,C testings on 4 different days. Four persons were tested in all combi- nations. As reference value for the relative response (RR), the response against one stimulator (a sup- posedly two haplotype response) is used. Some of the combinations are known to be weakly positive and this may explain the great variance in the experiment. The variance is at about the same level when using lo5 and 3 x lo5 stimulating cells.

expressed as cpm’s, Increments, SR’s, log SR and RR’s.

Kinet ics of responsiveness in MLC (Work- shop protocol part I I ) . Fig, 4 shows the cpm in an MLC reaction including zero, one and two haplotype disparities respec-

tively. The cultures have been harvested at days 3, 4, 5 and 6. An exponential growth has been found in all the one and two haplotype disparate combinations in- vestigated in this respect, but in identical combinations and autologous cultures the growth tends to be linear.

REPORTS FROM A MLC WORKSHOP 501

Figure 4. The cpm of one responder giving a “typical” zero, one, and two haplotype response. The cultures have been harvested on 4 different days. The growth is exponential in the one and two haplotype combinations. The haplotype disparity is indicated on the Fig.; auto indicates the autologous culture ( AAm) .

Analysis of f ami l y data ( W o r k s h o p proto- log converted abscissa, the cumulative col part 111). In Fig. 5, the results from percentage of the observations on the 983 one-way MLC tests are plotted on log- ordinate. The three groups of zero, one probit paper. The tests were performed in and two haplotype differences are each the period from April 1972 to April 1974. located on a straight line, indicating a The stimulation ratios are indicated on the normal distribution of the log SR within

502 REPORTS FROM AN MLC WORKSHOP

S R I I I I I I I l l

1 2 3 4 5 10 ;*so

Figure 5. Results of 983 one-way MLC tests in combinations of zero, one and two haplotype differences (filled in circles). The stimulation ratio is in- dicated on the log converted abscissa. The ordinate is the cumulative percen- tage of the observations. Straight lines indicate a normal distribution of the results. The open circles represent 12 different testings of one combination giving a typing response, the triangles 59 tests in 20 different combinations between five unrelated persons giving a mutual weakly positive response (tested 2-4 times).

each group. The mean and 95 % con- fidence value (t 2 sd) within each group are indicated in Table 3. There is consider- able overlapping between the one and two haplotype disparate combinations, but the average of the two haplotype different combinations is approximately twice that of the average of the one haplotype dif- ferent combinations when the average is based on calculations with log converted stimulation ratios. The lines defined by the open triangles and circles are examples of weak response and typing response (see later).

Weak response. The normal distribution of the three different groups of reactivity in MLC makes it possible, by repeated test-

ings, to define a given combination within one of these groups as described by Jersild

Table 3 Resirbs of 983 one way MLC tests,

April 1972-April 1974

Haplotype differences

Mean 12 6.5 0.95 Mean + 2 x s d 3.7 2.0 0.45 Mean + 2r:sd 38 21 2.0

The mean and 95$4 confidence level ( * 2 sd) for the three different groups of the normal ma- terial, indicated in Fig. 5 (filled in circles). The calculations are based on log converted stimula- tion ratios and the values retransformed into sti- mulation ratios.

REPORTS FROM A M I L WORKSHOP 503

et al. (1973). Some unrelated combina- tions only express a very weak MLC re- activity pattern. From a group of un- related persons, identical for HL-A1,8, and W15, five individuals were selected showing this weak MLC reactivity when tested repeatedly against each other. The results of 59 one way MLC testings (20 different combinations) are shown in Fig. 5 (triangles).

This grsup of weakly positive MLC re- actions is located between the group of identical siblings and the group of one haplotype different individuals. The same weak positive response is seen in siblings, identical for the strong LD locus but dif- fering for one or more of the SD antigens (Dupont et al. 1974). As some of the un- related weakly positive MLC combinations mentioned above differed by one LA anti- gen, is seems that a search for weakly posi- tive MLC combinations among unrelated should include not only HL-A identical combinations but even combinations iden- tical only for the FOUR locus.

Typing response. If an individual is homo- zygous for the SD and LD loci, it is pos- sible to use his cells as stimulating cells in an MLC test in order to characterize other individuals. If their lymphocytes do not respond to the test cells in an MLC test, they must carry the same MLC determi- nant as the stimulating test cell. Fig. 5 (open circles) shows the results of one MLC combination tested on 12 different days. An LD-7a homozygous test cell is used as the stimulating cell. The weakly positive response is very consistent and by repeated testings can be clearly distin- guished from the average of the group of HL-A identical siblings and of one HL-A haplotype differences. In other MLC com- binations, however, the cell can provoke strong responses, when used as stimulator, indicating that the responder cells do not

carry the same MLC determinant. When the weak MLC reactivity pattern is found by the use of SD and LD homozygous test cells, this response is called a typing re- sponse for the LD determinant defined by this test cell.

Discussion Investigation of the influence of irradia- tion on the responding and stimulating capacity of cells in MLC (Fig. 2) revealed that the responding capacity in MLC is severely inhibited already at a dose of approximately 500 rad, whereas the sti- mulating capacity is not blocked even at a dose of 5,000 rad. The MLC response in the cultures is very similar when mito- mycin-blocked cells and cells irradiated at our usual dose of 2,000 rad are used as stimulators.

The influence of thymidine concentra- tion in the medium is illustrated in Fig. 1. The usual concentration of 0.1 uCi per ml is not located on the steep part of the curve, but in order to fully saturate the system a tenfold increase would be needed. This would be rather expensive, but that could be circumvented by adding cold thy- midine (Schellekens & Eijsvoogel 1968).

The culture period is of great impor- tance to the amount of 14C incorporated. Our culture system allows a fully exponen- tial growth until at least day 6 (Fig. 4 ) . We do not know whether non-specific blastogenic factors will cause more ‘‘noise” in the system during a long culture period, which could make detection of typing re- sponses more difficult.

The variability of the system is caused by either technical or biological variations. The technical variations within one day seem to be of minor importance, as judged by the split experiment (Table 1) . In some cases, only repeated testings of the same combination can secure a safe interpreta- tion of the results. This is especially true

504 REPORTS FROM AN M I X WORKSHOP

in cases with weak reactions. In our hands, the most sensitive indicator of a weak re- sponse is possibly the stimulation ratio, whereas other expressions of the results might be more effective in the strong posi- tive reactions. As shown in Table 2b, the coefficients of variation in repeated test- ings are lowest when the data are expres- sed as log ratios. The normal distribution of this parameter within the different groups of MLC reactivity makes its use very convenient. We have tried to convert our results in the normal material to incre- ments or RR but found the variance greater. Moreover, the data expressed in this way were not normally distributed even if log conversion was tried. I t should be stressed, however, that the reference value was often ascertained by one un- related stimulator only.

The reproducibility of the relative re- sponse is influenced greatly by the re- ference value which must be well estab- lished. The use of a pool of stimulator cells, as described by Osoba & Falk (1974), may solve this problem by stabi- lizing the reference value. For some time we have used such a pool in our laboratory chosen from three individuals sharing no SD and possibly no LD determinants, as judged by MLG tests. By using this pool as reference, we have MLC typed patients with juvenile diabetes mellitus or with idiopathic Addison’s disease and found a striking and highly significant increase of the LD determinant 8a, which is the most common LD determinant linked to HL-A8 (Thomsen et al. 1974a).

In the definition of the typing response, one of the critical problems is the selection of the appropriate typing cell. Our search for such cells is primarily directed against the common LD determinants in the pop- ulation. Having access to a large panel of HL-A typed individuals, two approaches can be made, both based on the linkage

disequilibrium between FOUR and MLC. The first is described by Dupont et al. (197313). If two parents share an HL-A haplotype, one fourth of the children will be H G A homozygous. The MLC testing within this family will then reveal the oc- currence of LD homozygous individuals. If such an individual stimulates his parents only weakly in MLC, it indicates that his cells can be used as typing cells. The best way of testing this is the heterozygous back cross test, i.e. testing the mother and father in MLG against their HL-A SD identical children, if such are available.

Another way of finding homozygous test cells is by doing chess-board MLC tests between FOUR locus homozygous unre- lated individuals (Thomsen et al. 1974b). For the more common LD types, 10-15 individuals should be included in order to find two identical LD homozygous typing cells with mutual non response. Naturally, these should be tested afterwards within the families to prove the true homozygos- ity.

Within the group of weakly positive re- actions, the typing response may be dif- ficult to detect by only one investigation, and repeated tests may be necessary to classify the cells.

The SR of a typing response is usually somewhat higher than the SR of other weakly positive responses (Fig. 5 ) and this is possibly explained by the release of non- specific blastogenic factors from the LD homozygous cell, as this cell recognizes the strong foreign LD determinants of the re- sponder-cell (J~rrgensen et al. 1974). This adds to the weak positivity caused by dif- ferences at the weak LD locus.

Although it may be difficult to obtain completely negative MLC responses in SD non-identical related or unrelated combi- nations, it seems possible to use the test in the selection of bone marrow donors for patients having no HL-A identical sib-

REPORTS FROM AN M1.C WORKSHOP 505

lings. The observation of successful graft- ing and immunological reconstitution in a severe case of combined immunodeficiency (Dupont et al. 1973a, Koch et al. 1973) using a maternal uncle as donor indicates the clinical usefulness of the MLC test. In this case, there was disparity for three SD antigens and we did not find completely negative MLC responses between donor and recipient but under the appropriate conditions this does not seem to play a major role.

In our opinion, the most important point of the clinical application of the MLC test is the further characterization of low level responses which can be accepted in bone marrow grafting. In this respect, we consider the effect of unspecific blasto- genic factors an undesirable “noise” in the system, and means of eliminating or de- creasing these factors should be further explored.

Acknowledgemen t s The help of cand. scient. Lars Ryder has been very valuable for the statistical evaluations. Dr. Per Platz is thanked for critical comments. The Workshop part of the work would have been impossible to perform without the assistance of our chief technician Bodil Jakobsen. I am also indebted to Kirsten Rasmussen for her help in collecting Workshop data.

Finally, the skilful secretarial assistance of Ingerlise Christensen, Elisabeth Andersen and Janne Pedersen is gratefully acknowledged.

References Bach, F. H. & Amos, D. B. (1967) Major histo-

compatibility in man. Science 156, 1506-1508. Dupont, B., Andersen, V., Ernst, P., Faber, V.,

Good, R. A., Hansen, G. S., Henriksen, K., Jensen, K., Juhl, F., Killmann, S. Aa., Koch, C., Muller-Berat, N., Park, B. H., Svejgaard, A., Thomsen, M. & Wiik, A. (1973a) Im- munologic reconstitution in severe combined immunodeficiency with HL-A incompatible bone marrow graft: Donor selection by mixed lymphocyte culture. Transplant. Proc. 5, 905- 908.

Dupont, B., Jersild, C., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A. (1973b) Typing for MLC determinants by means of LD-homozygous and LD-heterozygous test cells. Transplant. Proc. 5, 1543-1549.

Dupont, B., Good, R. A., Hansen, G. S., Jersild, C., Nielsen, L. S., Park, B. H., Svejgaard, A., Thomsen, M. & Yunis, E. (1974) Two sepa- rate genes controlling stimulation in mixed lymphocyte reaction in man. PTOC. nat. Acad. Sci. (Wash.) 71, 52-56.

Jersild, C., Dupont, B., Fog, T., Hansen, G. S., Nielsen, L. S., Thomsen, M. & Svejgaard, A. ( 1973) Histocompatibility-linked immune- response determinants in multiple sclerosis. Transplant. Proc. 5, 1791-1796.

Jsrgensen, F., Lamm, L. U. & Kissmeyer-Niel- sen, F. (1974) MLC typing and blastogenic factor (abstract). Tissue Antigens 4, 404.

Koch, C., Henriksen, K., Juhl, F., Andersen, V., Dupont, B., Ernst, P., Good, R. A., Hansen, G. S., Jensen, K., Killmann, S. Aa., Muller- Berat, N., Svejgaard, A,, Thomsen, M., Wiik, A. & Faber, V. (1973) Bone-marrow trans- plantation from an HL-A non-identical but MLC-identical donor. Lancet i, 1146.

Mempel, W., Grosse-Wilde, H., Albert, E. & Thierfelder, S. (1973) Atypical MLC reac- tions in HL-A typed related and unrelated pairs. Transplant. Proc. 5, 401-408.

Osoba, D. & Falk, J. (1974) The mixed-leuko- cyte reaction in man: effect of pools of sti- mulating cells selected on the basis of cross- reacting HL-A specificities. Cell. Imrnunol.

van Rood, J. J . & Eijsvoogel, V. P. (1970) HL-A identical phenotypes and genotypes in unrelated individuals. Lancet i, 698-700.

Schellekens, P. Th. A. & Eijsvoogel, V. P. ( 1968) Lymphocyte transformation in vitro. I. Tissue culture conditions and quantitative measurements. Clin. exp. Zmmunol. 3, 571- 584.

Serensen, S.F. & Nielsen, L. S. (1970) The genetic basis for reactivity in human mixed lymphocyte culture. Acta path. microbiol. scand. 78, 719-725.

Thomsen, M., Christy, M., Lyngsee, J., Nerup, J., Ortved Andersen, O., Blatz, P., Poulsen, J. E., Rasmussen, K., Ryder, L. P., Staub Nielsen, L. & Svejgaard, A. (1974a) MLC typing in juvenile diabetes mellitus and idio- pathic Addison’s disease. Transplant. Rev. (in press).

Thomsen, M., Dupont, B., Jersild, C., Hansen,

10, 117- .

506 REPORTS FROM AN M1.C WORKSHOP

G. S., Nielsen, L. S., Platz, P., Ryder, L. P. & Svejgaard, A. ( 1974b) Linkage disequilibrium within the major histocompatibility complex in man (abstract). Tissue Antigens 4, 400.

Thorsby, E., Ria du Bois, Bondevik, H., Dupont, B., Eijsvoogel, V., Hansen, J. L4., Jersild, C., Jergensen, F., Kissmeyer-Nielsen, F., Lamm, L. U., Schellekens, P. Th. A., Svejgaard, A. & Thomsen, M. (1974) Joint Report from a mixed lymphocyte culture workshop. Tissue Antigens 4, 507-525.

Wood, N., Bashir, H., Creally, J., Amos, D. B.

& Yunis, E. (1972) A simple method of freezing and storing live lymphocytes. Tissue Antigens 2, 27-31.

Address : Mogens Thomsen Tissue Typing Laboratory Blood Bank and Blood Grouping Department Rigshospitalet Copenhagen Denmark