Human HL-A transplantation antigens: Separation of molecules carrying different immunological specificities determined by a single genotype

Vol. 33, No. 1, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

HUMAN HL-A TRANSPLANTATION ANTIGENS: SEPARATION OF MOLEKXrLESCARRYING DIFFERENT IMMUNOLOCICAL SPFXXFlCITIES DETERMINED BY D. A. L. Davies,

J. Colombani,

D. C. Viza,*

SINGLE GENOTYPE

A

and J. Dausset

Searle Research Laboratories, High Wycombe, England and Institut de Recherche sur les Maladies du Sang, Hepita St. Louis, Paris, France.

ReceivedAugust19,

1968

The HL-A genetic a primary In this arated

role

locus

in tissue

graft

paper we show that by

DEAE-Sephsdex

immunological

For H-2 antigens sygous animals)

determinants

and its

fractions

that

individual

carrying

phenotypic

products

and the H-2 transplantation

autolytically

play

can be sep-

different

antigens

retarded

HL-A

1967).

methods but yields for detailed

excluded

Further

are very analysis.

is not yet known in what part

The soluble

bromelin

or

preparations, fractions

can be achieved

small and insufficient

by

pure material

The best preperatione of the molecules

1968%).

can be solubil-

and included

purification

similar

5 to 14 in homo-

of the enzymes ficin,

1966; Davies,

fraction.

(from

(Davies,

H-2 antigens

or by the addition

and Davies,

are remarkably

a selection

G2OG Sephadex, give H-2 inactive

has been obtained but it

different

from a single

of about 28 known specificities.

and an H-2 active

teins

sntigene

antigens

Hamburger and Mathe', 1968).

(Dausset,

each genotype determines

papain (Nathenson

conventional

leucocyte

determinants.

to the mouse H-2 locus

run through

rejection

HL-A

into

This complex locus

ised either

in man determines

are glycopro-

the immunological

reside.

Human HL-A antigens

* Attache/ de recherohe

have been found to behave in the same way as mouBe

‘B 1'I.N.S.E.R.M.

88

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H-2 antigens, the degree of similarity genetic homolgoues (Davies, Viza, stone, Viza,

suggesting that they are likely

Colombani and Dausset, 1967; Davies, Man-

Colombani and Dausset, 1968).

someH-2 specificities

to be

Mouse studies have revealed that

can be separated from one another by gel filtration

(Shimada and Nathenson, 1967) and also by DEA?Iion exchange chromatography (Davies, 1968b_). By the latter

method nearly all

of the eleven H-2 antigens

of one mouseH-2 genotype can be separated (Summerell and Davies, 1968).

MATWIALS AND MMTHODS Fresh normal humanspleens were used and antigens were extracted solubilized

as already described (Davies, 1966; Davies et.

results below were obtained with autolytic erial

preparations.

1968).

The

Crude soluble mat-

was passed through G200 Sephsdex and the retarded fraction

centrated

and

pooled, con-

in the cold under reduced pressure, dialysed and freeze dried.

For

examination on DEAEcolumns, material was dissolved in and dialysed against starting

buffer

(0.05 M Tris,

pH8) and in the case quoted (spleen number 18)

59 mg in 10 ml was placed on a column 125 cm x run to 0.05 M Tris, 5

ml

4.5

fractions ml

cm. A linear

pH9 + 0.35 M NaCl over a volume of 1000 ml.

were collected,

of each freeze dried.

physiological

1.5

salt solution

of cytotoxicity

These were reconstituted

in 0.9

ml

1968).

tion were tested; each fraction

of buffered

and stored at -70'.

induced by isoantisera

blue exclusion method, or by inhibition (Davies et.

Two hundred

each dialysed against 0.01 M NH4HC03and

Fractions were tested for HL-A immunological specificities inhibition

gradient was

of platelet

For the latter,

for inhibition

serial

and measuredby the trypan complement fixation

dilutions

of the cytotoxic

either by

of each column frac-

reaction

a fixed dose of

was used and the extent of combination with antibody assessed

as percentage of surviving

target

cells

89

(peripheral

lymphocytes).

Vol. 33, No. 1, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The spleen donor was typed for HL-A specificities *1

3

4

5

6

7

10

8

-+-++++-

as follows:

11

12

14

16

-

-

-

+

RESULTS The results clarity.

exe shown in Fig. 1, separated into parts (a) and (b) for

For economy, regions of the columns were checked where mouseH-2

antigens would have been expected if H-2 material had been run. the positions

of the HL-A antigens in the predicted places and all

tions were then tested to confirm negative reactions the clear separation of two antigens, serum Wo"),

coincident

other fracFSg. 1% shows

HL-A.16 and one unknown (detected with

the two peaks being centred on tubes 131 and 104 respectively.

These positions Specificity

elsewhere.

This revealed

are reflected

5 (an allele

in a more complex fashion in Fig. lg.

of the HL-A second sublocus) appears mainly in a peak

with HL-A.16 (en allele

of the HL-A first

sublocus) but with a

smaller peak at tube 104; there is also a possible smaller peak at 113 (but one tube only) and a shoulder at tubes 117-121. specificity,

('*Juivt),

but in addition,

Another serum of unknown HL-A

also shows a major component coincident

with antigen 16

may have lesser components corresponding to the minor compon-

ents shownby the specificity tested qualitatively,

Reaction with antiserum to HL-A.6,

is shown by the shaded area and again covers

both main antigen positions, as for the other specificities a serum anti HL-A.l,

5 reaction.

the area of

being negative from tubes 1 to 95 and 141 to 200 Tubes were tested for reactivity

tested.

a specificity

with

the donor did not possess, and no reactions

were found.

* Nomenclature is that of Dausset, Ivanyi, Colombani, Peingold and Legrand (1967). Two of the antisera for which there are alternative terms are: GA.5 = 4' (Payne) = To.5 (Ceppellini) = 6 (Terasaki). HL-A.16 = Lc.11 (Walford).

90

Vol. 33, No. 1, 1968

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

l-

a 30- LI

-51

- 6(

-7(

b

i

I

I

100 TUBE

1

110 120 NUMBERS

0 J20:

I

130

Fig. 1. Human soluble HL-A transplantation antigen examined on DEB-Sephadex A50 (linear salt gradient), center section shown. Tube numbers are for 5 ml Tests for different HGA specificities of the original spleen fractions. donor have been plotted on arbitrary scales. (a) Reaction with serum “MO”, Reaction with serum anti-HL-A.16, W V, (cytotoxicity inhibition). (platelet complement fixation inhibition). Shaded area is the extent of qualitative reaction with serum anti-I&-A.6, (inhibition of oytotoxioity). (b) Reaction with serum anti-HL-A.5, o----O, and reaction with HL-A antiserum rrJuill , W, (both by platelet complement fixation inhibition). Protein profile (280 w absorbence), dotted line. The protein Rig. li

profile

from tubes

the antigen

positions

(by 280 w absorption)

80 to 155.

There are protein

in the region

of serological

91

is shown by the dotted peaks coinciding reactivity.

with

line

in

each of

Vol.

33,No.

1, 1968

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISCUSSION AND CONCLUSIONS The separation

of different

HL-A specificities

DEAE columns enhances the idea of genetic Two main positions with

serum anti

are indicated

HL-A.5.

positions

but exact coincidence

ificities

of a given H-2 genotype

The interpretation

monospecific.

e.g.

(Summerell

usually

all

almost

with

means that

is that few of the available

Antiserum HL-A.6 reacts over

on

one of these spec-

and Davies,

by a reaction

monospecific and the HL-A.5 antiserum used may recognise antigen.

with

In the mouse

are separable

In the mouse this

positions

the mouse H-2 system.

peaks appear to coincide

peaks revealed

The probability

separate

by the two components detected

is not claimed.

of multiple

serum is not yet clear.

homology with

so far,

The other

into

more

1968).

a single

anti-

the serum is not

HL-A antisera are than one HL-A

the extent of both positions defined in

Fig. l_a and might have resolved into two (or more) peaks if it had been possible to test dilutions

of each fraction

in that

system.

Correspondence of protein and antigen peaks is suggestive that the antigens themselves may be revealed in the protein ature to attach significance

to this until

Failure of any fractions as a specificity

control;

more

profile

but it would be prem-

data have accumulated.

to. react with the antibody HL -A.1 (M ac) serves

antigen 1 was not present in the original

donor, but only other alleles

of the HL-A first

spleen

sublocus (e.g. HL-A-16).

There is no suggestion thus far that the two main peaks represent separations of antigens determined by the two main HL-A subloci that have recently

been

defined (Dausset, Walford, Colombani, Legrand, Feingold and Rapaport, 1968). The importance of establishing systems

the similarity

of humanHL-A and

mouse

is that experimental work can be carried out in the mousewhere human

studies are impossible and the importance of humantissue transplantation quires the information

that

mice

are separable may have different cell stimulation tolerance

H-2

for.matching

may

possibly provide.

biological (Viza,

properties

Thus antigens that e.g. of immunogenicity,

Degani, Dausset, and Davies, 1968)

induction.

92

re-

or

Vol. 33, No. 1, 1968

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENTS It

is a pleasure to thank our assistants

J. Manstone, Mr. V. Baugh, Miss Barbara Alkins, Degani, Mlle. Catherine Dehay and Mrs. Christine

for excellent

support, m. A.

Mrs. Sue Buckham, Mile. Ora Brittin.

REFERENCES Dausset, J., Hamburger, J. and Mathg, G., '*Advance in Transplantation*', Munksgaard, Copenhagen, (1968). Dausset, J., Ivanyi, P., Colombani, .I., Feingold, J. and Legrand, L., in "Histocompatibility Testing 1967", ed. Curtoni, E. S., Mattiuz, P. L. and Tozi, R. M. p. 194. Munksgaard, Copenhagen(1967). Dausset, J., Walford, R. L., Colombani, J., Legrand, L., Feingold, N. and Rapaport, F. T., 2nd Int. Congr. Transplantation Sot., New York, in press (1968).

Davies, D. A. L., Immunology, 11, 115 (1966). 2, 31 (1967). Davies, D. A. L., Transplantat&, ed. Rapaport, F. T. and Dausset, Davies, D. A. L., in "HumanTransplantation'*, J. Chap. 38. Grune and Stratton, New York (1968~). Davies, D. A. L., in "Advance in Transplantation", ed. Dausset, J., Hamburger, 5. and Mathe, G. p. 275. Munksgaard, Copenhagen(1968k). Davies, D. A. L., Colombani, J., Viza, D. C. and Dausset, J., in "Histocompatibility Testing 1967", ed. Curtoni, E. S., Mattiuz, P. L., and Tozi, R. M. p. 287, Munksgaard, Copenhagen(1967). Davies, D. A. L., Manstone, A. J., Viza, D. C., Colombani, J. and Dausset, J., Transplantation, 6, No. 4 (1968). Nathenson, S. G. and Davies, D. A. L., Ann. N.Y. Acad. Sci., 2, 6 (1966); Proo. Nat. Acad. Sci., 2, 476 (1966). Shimada, A. and Nathenson, S. G., Biochem. Biophys. Res. Comm., 29, 828 (1967). Summerell, J. M. and Davies, D. A. L., 2nd Int. Congr. Transplantation Sot., New York, in press (1968). Viza, D. C., Degani, O., Dausset, J. and Davies, D. A. L., Nature, 2, 704

(1968).

93