Transplantation antigens in a high molecular weight form—II Non H-2 mouse antigens and membrane structure

lmmunochemistry. Pergamon Press 1971. Vol.8, pp. 17-23. Printedin Great Britain

T R A N S P L A N T A T I O N A N T I G E N S IN A H I G H MOLECULAR WEIGHT FORM--II N O N H-2 M O U S E A N T I G E N S A N D MEMBRANE STRUCTURE D. A. L. DAVIES, U. H)~MMERLING* and BARBARA J. ALKINS Searle Research Laboratories, High Wycombe, England, and Institute of Virology, University of Giessen, West Germany

(First received 11 May 1970; in revisedform 29July 1970) A b s t r a c t - Mouse thymus cell membrane preparations were treated with sodium dodecyl-

sulphate and starch stearate and the soluble fraction was examined on DEAE Sephadex. One component carried H-2 antigen with Ly-A. Ly-B and theta antigens: other components carried theta antigen only. TL antigens were present but could not be exactly located in relation to the other alloantigens. The preparations are discussed in the light of membrane structure. INTRODUCTION T h e external organization o f microbial cells is r a t h e r well u n d e r s t o o d and in a general way the plasma m e m b r a n e , cell wall and the exposed surface coverages are broadly similar in their structural a r r a n g e m e n t s a m o n g bacteria. T h e functional roles are also becoming clarified, not only in relation to the external env i r o n m e n t but also internally, e.g. with respect to e n z y m e content and the anchorage point for the chromosome. Mammalian cell m e m b r a n e s are much m o r e poorly understood. A division o f labour may be expected between nuclear and plasma membranes, when c o m p a r e d with the bacterial situation. With r e g a r d to the plasma m e m b r a n e , the trend o f interest passes f r o m the many lipid-protein models a m o n g which we may exercise o u r choice and which might be r e g a r d e d as the grass, to the elaborate covering, the flowers, that has been studied to some extent by purely chemical m e t h o d s (with no definitive results so far) and also by immunological methods. O n the o t h e r h a n d there may be no grass; the functional units and c o m p o n e n t s recognized by antigenic characteristics may themselves provide the structure, in which case the protein o f the physico-chemical models would be heterogeneous. No role can clearly be attributed as yet to those surface-located substances that are called antigens, so n a m e d only because antibodies are used to detect them. Nevertheless isolation and characterization o f these substances is most likely to lead to clues as to their role. T h e greatest d e g r e e o f i m m u n o genetic discrimination can be obtained in allogeneic situations where gross species differences are excluded. Alloantigens have been most studied on red cells but these are o f a cell type too specialized and unrepresentative to provide data f r o m which one would dare to generalize. Alloantigens o f leucocytes are increasingly falling within the *This work was initiated while U.H. worked at the Sloan-Kettering Institute for Cancer Research, New York. 17

I.M.M. Vol. 8 No. 1 --B

18

D. A. L. DAVIES, U. HAMMERLING and B. J. ALKINS

sphere of practical study and a sketchy picture of their topographical relationships is now building up; no structural unit has been found into which the various alloantigens can be fitted, but a precise and purposeful arrangement is suggested from various lines of study and indicated rather directly by the mapping studies of mouse thymocytes [ 1] and by immuno-ferritin methods [2]. In the mouse the major histocompatibility antigens (H-2) are being studied in soluble form; gentle proteolysis yields a family of molecules, of molecular weight mainly in the region of 50,000 [3] each molecule carying usually only one or a few of the specificities determined by any particular H-2 genotype [4]. The same is true for HL-A antigens of human lymphocytes[5]. This implies some nonrandom degradation of a larger organized molecular complex. TL (thymusleukaemia) is another (non H-2) alloantigen which can be solubilized enzymically and separated from H-2 antigen by DEAE ion exchange chromatography [6]. The preceding paper[7] describes a method for preparing, from mouse spleen cells, a much larger soluble membrane product that probably carries all of the H-2 alloantigenic specificities of the cell. The same procedure provides HL-A antigen in high molecular weight form from human leucocytes[8]. In this paper similar extracts of mouse thymocytes have been examined for their content of H-2 antigen and of the non H-2 alloantigens, Ly-A, Ly-B,[9] theta [10] and TL[ll]. MATERIALS AND METHODS Mice

Spleens and thymuses were removed from mice of strain 129(H-2 h, TL-2, Ly-A.2, Ly-B.2. 0c3n) and disaggregated into cell suspensions. Thymuses were used as the source of TL-2 antigen that does not occur elsewhere, except on leukaemic cells. Batches of 300 thymuses were used for most preparations.

A Blige~t Thymus cell suspension was eluted twice with hypotonic NaC1 and crude membrane-derived material deposited from these cell washings by centrifugation at 30,000 rev/min (30 rotor, Spinco L-2-65) for 2 hr. This product was stored at 4° in aqueous suspension with thymol as preservative. This procedure has previously been described in detail[12]. In a typical extraction the product was 223 mg (dry wt.). This was centrifuged at 40,000 rev/min (40 rotor, Spinco L2.65) for 2 hr to remove any residual soluble material and half of the sedimented fraction (46 mg) used in the first experiment described below. The material was resuspended in 20 ml of 0-3 per cent sodium dodecylsulphate (SDS) and 1 mg/ml starch stearate (SST) at 4°. The preparation of starch stearate has been described by H~immerling and Westphal[13]. After centrifugation at 40,000 rev/min (Spinco L2"65) for 2 hr the supernatant was dialysed to remove SDS and freeze dried. This weighed 42 mg and allowing for the content of SST, represented 22 mg of membrane derived soluble fraction, the insoluble residue weighed 20 mg. From the soluble fraction a sample (2 mg) was put aside for assessment of antigen content and the remainder (40 mg) dialysed against starting buffer (0.05 M Tris pH 8.0) and loaded on to a DEAE-Sephadex A50 column. This

Mouse Non-H-2 Antigens

19

was eluted with a linear gradient from this buffer to 0.5 M (0.05 MTris pH 9.0 + 0.45 M NaC1), under conditions previously described for separating the different components of mouse H-2 alloantigens [4] and human HL-A transplantation alloantigens [5]. The fractions collected (150 × 2 ml) were first used to measure 280 m/~ absorbance and conductivity and were then individually dialysed against 0-01 M NH4HCO3, freeze dried and each reconstituted in 0.5 ml barbiturate buffered saline and stored at - 70° to await serological examination.

Serology and antisera Antigens were detected in column eluate fractions by single point assays and the activity of other solutions by dilution assays, using their ability to specifically inhibit the cytotoxic effect of suitable alloantisera upon appropriate target cells in excess complement (guinea pig serum). Such assays have been described previously [4]. Systems defining the antigens considered below are detailed in Table 1.

DEAE ion exchangechromatography This was carried out as described by H~immerling, Davies and Manstone [7]. RESULTS Serological analysis of a DEAE column run using strain 129 thymus derived material is shown in Fig. l(a). H-2 antigen was measured as H-2-5. Antigens Ly-A.2 and Ly-B.2 were located in the H-2 region but tests were inadequate to show the exact peak positions in this case. Theta antigen showed three peaks, the first coinciding with the main H-2 antigen component, the second close to a shoulder of H-2 reactivity and the third not associated with any other alloantigen measured. This general picture was confirmed but not clarified using a preparation from BALB/c mice (H-2 d, TL-2, Ly-A.2, Ly-B.2, 0c:~u). A preparation made from "~ 40

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AKR (H-2 k, 0 AK~) C57BL/6 (H-2 b, TL-) CE(H-2 k, Ly-A.2, Ly-B.1) DBA/2 (H-2 d Ly-A. 1. Ly-B.1) C57BL/6 (H-2 k Ly-A.2, Ly-B.2) C3H × I (H-2 k Ly-A.2, Ly-B.1)

A/J (H-2 ~, 0c:~H)

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CE (H-2 k, Ly-A.2 Ly-B.1)

EL4 (H-2 b, Ly-A.2, Ly-B.2)

H-2-2,6,14 etc. Ly-B.2

Ly-B. 1

H-2.2.5 etc. Ly-A.2, Ly-B.2

Ly-A.2

H-2-1.3.4 etc. TL- 1,2,3

H-2.4,6.10 etc. 0c:m, Ly-B. 1

H-2.2,5,22,33 Ly-A.2

Relevant antibodies expected

BI0.Br (H-2 k Ly-A.2, Ly-B.2

DBA/2 (H-2 d, Ly-A.1, Ly-B.1)

BALB/c (H-2 d, Ly-A.2, Ly-B.2)

DBA/2 (H-2 d, Ly-A.1, Ly-B.1)

ERLD (H-2 b, TL- 1,2,4)

C3H (H-2 k, 0c:3a), Ly-B. 1

AKR lymphocytes (H-2 k)

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All Ly alloantisera were kindly provided by Drs. E. A. Boyse and L. J..Old, of the Sloan-Kettering Institute for Cancer Research, New York. All Ly cell d o n o r strains were T L neganve and 0 c3H, and Ly serum producers 0c3H. *Thymocytes were used except where leukaemias (ASL1, ERLD and EL4) or lymphocytes are indicated. tTL-2 antibody not detected at the serum dilution used. ~Ly-A.2 antibody not detected at the serum dilution used.

DBA/2 (H-2 d)

Recipient mouse

EL4 (H-2 b)

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Table 1. Antisera and test systems

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21

Mouse Non-H-2 Antigens

an H-2 a strain lymphoma (RADA-2, since lost) revealed that TL-1 antigen was present in one of a series of pools made from a slightly different gradient elution; H-2 antigen was confined to the same pool. A subsequent run, again using strain 129 thymus cells, clarified the position of the Ly-A and Ly-B antigens, as shown in Fig. l(b), where they are seen to coincident with H-2. The TL-2 antigen was particularly difficult to find but average results from several attempts at the limit of sensitivity of the tests indicated the same position as H-2 with some reactivity extending on into the region of theta reactivity. A pool made over the 'Ly region' of the column shown in Fig. l(a) (tubes 49 to 75) was recovered and tested by inhibition in Ly systems with results shown in Figs. 2 and 3. Unlike any other isoantigens studied, the products were reactive in both systems, i.e. Ly-A.2 material reacted to a considerable extent in the LyA.1 system, and Ly-B.2 material was no more reactive in its own specific system than in an Ly-B. 1 system.

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Fig. l(b). A similar experiment to that of Fig. l(a), but showing Ly-A and Ly-B reactions. H-2, © ©; Ly-A.1, 0 - - - - 0 ; Ly-A.2, & A. DISCUSSION In the light of previous studies of soluble H-2 antigens prepared by proteolysis and separable from each other by DEAE chromatography, the fact that SST solubilized antigen does not resolve in this way suggested that many H-2 specificities reside on one molecular complex[7]. Such a complex would be a likely source of the smaller fragments of H-2 antigen of molecular weight about 50,000. At this level of membrane disassembly H-2 antigen is a glycoprotein; theta antigen has protein properties [14]. That the material being studied is not soluble antigen released autolytically and subsequently adsorbed onto starch stearate is shown by the failure of H-2 soluble antigen to adsorb onto starch stearate [7], and in any case theta and Ly antigens have not previously been obtained in soluble form. The present results

22

D.A.L.

DAVIES, U. H A M M E R L I N G and B. J. A L K I N S

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Fig. 2. Material pooled from the column illustrated in Fig. 1 (tubes 49-75 from the region of Ly antigen elution. Doubling dilutions started from a solution at 3 mg/ml, serum titres shown by broken lines and serum used for inhibition tests at 1/240 (Ly-A.1) and 1/60 (Ly-A.2), separate scales are shown for the two systems. Material extracted from BP8 (H-2 k) ascites sarcoma cells and used as a specificity control, showed no inhibition in either system, being an Ly negative cell (Dr. Boyse, personal communication).

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Fig. 3. A test similar to that shown in Fig. 2, but in the systems Ly-B. 1 and LyB.2, with BP8 material as specificity control. Sera used at 1/42 (Ly-B.1) and 1/108 (Ly-B.2) that showed closely similar sensitivity points on the titre checks (broken lines).

Mouse Non-H-2 Antigens

23

thus suggest that the molecular complex probably carries not only multiple H-2 specificities but also determinants of other alloantigens, TL, Ly-A, Ly-B and theta, although the possibility of f u r t h e r resolution is being studied. In fact it is unlikely that the region of reactivity for H-2 + Ly-A + Ly-B + T L on the columns represents a single molecular species because Ly-A and Ly-B have been shown to occupy remote sites on the cell membranes [1]. However, one fraction of theta antigen can be seen free from the other alloantigens following DEAE chromatography. If the SST derived antigen represented a unit of surface m e m b r a n e structure this would be expected since the thymocyte surface m a p p i n g data[l] show a close associated of some of the alloantigens, but theta antigen is more widely dispersed and occurs both close to and some distance away from H-2 and T L and Ly-A (or Ly-B). T h e small yield of SST soluble antigen obtained thus far will have to be improved before the m e m b r a n e unit suggestion can carry much weight but it should be recalled that yields of all soluble forms of leucocyte alloantigens obtained up to the present time by any method have been very small. T h e feature of Ly-A.1/A.2 and Ly-B.1/B.2 cross reactivity has been characteristic of these antigens in earlier studies [Davies, unpublished] and has not been observed in other systems, e.g. H-2, HL-A, TL, or even in other simple two allele-systems such as theta. T h e finding is that at the intact cell level good specificity is f o u n d for Ly-A.1/A.2 and Ly-B. 1/B.2 but after extraction by any means, specificity is lost. One possibility that suggests itself is that the basis of the difference, Ly-A. 1 to Ly-A.2 (of Ly-B. 1 to Ly-B.2) is one of orientation of molecules in one way or another such that both specificities are present on the molecule but only one is surface exposed, d e p e n d i n g on the Ly-A and Ly-B genotypes. A precedent for this was put forward previously for a species specific mouse antigen [ 15]. Acknowledgements-We are indebted to Mr. A.J. Manstone, Mr. V. S. G. Baugh and Mrs.

Christine Brittin for technical assistance. REFERENCES 1. Boyse E. A., Old L.J. and Stockert E., Proc. natn. Acad. Sci. U.S.A. 60, 886 (1968). 2. Aoki T., H~imrnerling U., de Harven E., Boyse E. A. and Old L.J.,J. exp. Med. 120, 979 (1969). 3. SummerellJ. M. and Davies D. A. L., Transplantation Proc. 1,479 (1969). 4. Davies D. A. L., Transplantation 8, 51 (1969). 5. Davies D. A. L., Colombani J., Viza D. C. and Dausset J., Biochem. biophys. Res. Commun. 33, 88 (1968). 6. Davies D. A. L., Alkins, B. J., Boyse E. A., Old L. J. and Stockert E., Immunology 16, 669 (1969). 7. H~immerling U., Davies D. A. L. and Manstone A. J., Immunochemistry 8, 7 (1971). 8. Davies D. A. L., Colombani J., Viza D. C. and H~immerling U., Clin. exp. Immunol. in press (1970). 9. Boyse E. A., Miyazawa M., Aoki T. and Old L.J., Proc. R. Soc. B170, 175 (1968). 10. ReifA. E. and AllenJ. M. v.,J. exp. Med. 120,413 (1964). I1. Boyse E. A., Stockert E. and Old L.J.,J. exp. Med. 128, 85 (1968), 12. Davies D. A. L., Immunology 11, 115 (1966). 13. H~immerling U. and Westphal O., Eur.J. Biochem. 1, 46 (1967). 14. O'Neill G. J., Reynolds B. L. and Davies D. A. L., to be published. 15. Boyle W. and Davies D. A. L., Immunology 11,353 (1966).