580

D.Snary, C.J. Barnstable, W.F. Bodmer and M.J. Oumpton

Eur. J. ImmunoL 1977.8: 580-585

D. Snary", C.J. Barnstable', W.F. BOdmer' and M.J. Crumpton'

Molecular structure of human histocompatibility antigens: the HLA-C series"

National Institute for Medical Research, London' and Genetics Laboratory, Department of Biochemistry, Oxford'

The HLA-CW2 antigen of the B lymphoblastoid cell line BRI 8 is structurally homologous to t h e HLA-A and B antigens as judged by various criteria. Each antigen comprised a glycosylated polypeptide of 43 000 rnolecular weight that is noncovalently associated with 02-microglobulin (Pz m). Some small differences in molecular parameters were, however, revealed. Thus, the deoxycholate-solubilized HLA-CW2 antigen sedimented at the same rate as the HLA-A antigens but at a slightly faster rate than the HLA-B antigens. This variation is apparently due to different amounts of bound deoxycholate. Also, whereas essentially all of the HLA-A and B antigens and about half of the HLA-CW2 antigen were adsorbed Rrongly b y Lens culinaris lectinSepharose, the remaining HLA-CW2 antigen was bound much more weakly and did not require sugar for elution. This difference reflects some structural heterogeneity in the carbohydrate moiety of the HLA-CW2 antigen. The results of various studies suggest that the HLA-CW2 antigen is expressed t o a lower extent than the HLA-A or B antigens and that essentially all of the P2m o f the BRI 8 plasma membrane is associated with the HLA-A, B and C alloantigenic polypeptides.

1. Introduction The expression of the classical transplantation antigens of man is controlled by the major histocompatibility gene system, the HLA complex. Initially two series of polymorphic cell surface antigens, the HLA-A and B series, were defined. These series of antigens have been extensively studied serologically and biochemically [ 1, 21. Each series comprises 1 8 t o 20 serologically defined variants which are expressed as if controlled by alleles at the corresponding loci, A and B. More recently, a third allelic series of antigens, H L A C (formerly designated AJ), has been described [ 3,4]. Serological studies of families in which intra-HLA recombination has occurred have placed t h e C locus between the A and B loci. The small number of recombinants so far examined suggest a distance of about 0.2 centimorgans between the B and C loci and about 0.6 centimorgans between the A and C loci [5].

b y a gene within t h e major histocompatibility complex on chromosome 6 [ 12, 131. In contrast, P2m is genetically unlinked to the HLA-A and B genes, being coded b y a gene on chromosome 15 [ 141. Recent studies have, however, shown that the genes are functionally related, since HLA-A and B antigen biosynthesis and expression o n the cell surface is dependent o n a functional flZ m [ 151. Much less information is available regarding the structure of the HLA-C antigens, although it is apparent from serological studies that the C series antigens are also associated with P2m [ 161, and have a similar tissue distribution t o the HLA-A and B antigens, being present on T and B lymphocytes and platelets but not o n erythrocytes [ 171. The present paper is concerned with the molecular properties of the HLA-C antigens and the comparison of their structure with those of the HLA-A and B antigens.

2. Materials and methods The gross molecular structures of the HLA-A and B antigens have been established [6, 71. A comparison of their N-terminal amino acid sequences clearly indicates that they arose by gene duplication [ 8- 1 11. They comprise a 43 000 molecular weight glycosylated polypeptide chain which is noncovalently bound t o a 12 000 molecular weight nonglycosylated polypeptide, namely Pz-microglobulin (&m). The larger chain carries the serologically detected polymorphism and is coded

[I 17251

* This work was supported in part by a grant from the Medical Research Council to the Genetics Laboratory. 0

Present address: Department of Immunochemistry, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, GB

Correspondence: Michael J. Crumpton, National Institute for Medical Research, Mill Hill, London NW7 lAA, GB Abbreviations: A m : &Microglobulin

SDC: Sodium deoxycholate

2.1. Lymphocyte plasma membrane

BRI 8, a human lymphoblastoid B cell line (HLA-A1, A2, B8, B13, CW2) grown by Searle Diagnostics (High Wycombe, Bucks., UK) was used as the source of HLA antigens. Plasma membrane vesicles were prepared as previously described [ 181. Briefly, viable cells ( 5 x 107/ml of 0. I5 M NaCl 10 mM Tris-HC1 buffer, pH 7.4) were broken using a Stansted Cell Disrupter (model no. A 0 612 with disrupting valve no. 516; Stansted Fluid Power, Stansted, Essex, UK).The plasma membrane fraction was separated from the cell homogenate by differential centrifugation and purified by sucrose density gradient centrifugation. The purified membrane was not contaminated t o a significant extent by other subcellular components as judged by various biochemical and morphological criteria. Plasma membrane ( 4 mg of protein/ml) was solubilized at 0 'C in 2 % (w/v) sodium deoxycholate (SDC) 10 mM TrisHCl buffer, pH 8.2, and insoluble material was removed by centrifuging at 100 000 x g for 1 h. Under these conditions, the supernatant contained 90 % of the membrane protein and more than 8 5 % of the HLA-A, B and C antigen activities.

Structure of HLA-C antigens

Eur. J. Immunol. 1977.8: 580-585

2.2. Antisera HLA-A, B and C antigens were detected using pregnancy sera and antisera produced by planned immunization that had been characterized relative to the specificities defined by the 5th and 6th Histocompatibility Testing Workshops [ 1, 191. The source and specificity of the sera are shown in Table 1. Antisera which possessed more than one specificity were rendered effectively monospecific by using appropriate target cells. Allogeneic (no. P3302B, P2604B and P1530B) and xenogeneic antisera against HLA-la antigens were described previously [ 201. The rabbit anti-human P2m serum employed for inhibition assays was kindly donated by Dr. Howard Grey. The goat antiserum against P2m was raised by immunization with a purified preparation of human urinary Pzm which gave one detectable band only of 12 000 molecular weight o n polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Each antiserum was judged t o be monospecific on the basis of its pattern of reactions with various human cell lines, including its lack of reactivity with Daudi and erythrocytes [ 131, and its inhibition by an authentic sample of &m. In addition, when tested on a panel of human-mouse somatic cell hybrids they reacted only with those cells possessing human chromosome 15. Table 1. Source and specificity of allogeneic antisera against HLA-A, Band C antigens Serum Number F1209D F1014H F3002X F5221B P1422B PI 0408 P4375B

Source

Specificity')

Name DOWY

Bodmer

Gillespie

Payne/Bodmer Bodmer/Payne/Hyland Cohen (RF'MI)

Caminiti Yochum

-

OPSb) OPS

OPS

Al, B8 A1 A2, A9

A2 B8 B13 cw2

a) As defined with respect to cells typed in the Vth and VIth Histocompatibility Testing Workshops. b) Oxford pregnancy serum.

2.3. Analytical methods Stoke's radii were determined by gel filtration on columns (85 x 1.6 cm) of Ultrogel AcA 34 (LKB Instruments Ltd., London), eluted with 0.5 % SDC 10 mM Tris-HCI buffer, pH 8.2, relative to the elution characteristics of water-soluble globular proteins of known Stoke's radii (aldolase, 4.5 nm; bovine serum albumin 3.55 nm; ovalbumin, 2.73 nm, soybean trypsin inhibitor, 2.36 nm). S z ~ , w values were estimated on sucrose density gradients of 8 t o 20 % (w/v) sucrose in 0.5 % SDC 10 mM Tris-HCI buffer, pH 8.2, centrifuged in a Beckman SW41 rotor at 38 000 rev/min for 24 h, relative to the rate of sedimentation of standard water-soluble globular proteins run at the same time (aldolase, 7.5 S; bovine serum albumin, 4.42 S; ovalbumin, 3.55 S; soybean trypsin inhibitor, 2.3 S). BRI 8 plasma membrane solubilized in 2 % SDC 10 mM Tris-HCl buffer, pH 8.2 (0.5 ml containing 2 mg of membrane protein) was used as the antigen source. HLA-A, B, C and la

581

antigens were located on gel filtration and sucrose density gradients by measuring antigenicity. Molecular weights were calculated from Stoke's radii and S20,w as described previously Clycoproteins were specifically adsorbed to columns ( 15 x 1.5 cm) of Lens culinnris (lentil) lectin-Sepharose 4B ( 1 mg of lectin/ml of gel sediment) in 0.5 % SDC 10 mM Tris-HC1 buffer, pH 8.2, and were subsequently eluted with 2 % methyla-Dmannopyranoside in 0.5 % SDC 10 mM Tris-HC1 buffer, pH 8.2 [221Pzm-associated components of SDC-solubilized BRI 8 plasma membrane (20 ml containing 16 mg of membrane protein) were specifically depleted by adsorption to Columns (20 x 1.5 cm) of the immunoglobulin fraction of a goat anti-p2m serum attached to Sepharose 4B (10 mg of Ig/ml of gel sediment). Columns were eluted with 0.5 % SDC 10 mM Tris-HCl buffer, pH 8.2, at a flow rate of 15 ml/h. 2.4. Antigenic activity

Antigenic activity was assessed quantitatively by inhibition of fluorochromatic microcytotoxicity as described previously [ 18, 231. Peripheral blood lymphocytes, separated from freshly drawn defibrinated blood by flotation on Ficoll-Triosil gradients, were used as target cells in assays for HLA-A, B and C antigens and &m. HLA-Ia antigens were assayed using cultured lymphoblastoid B cell lines. Rabbit complement for use in the latter assay was selected for lack of naturally occurring heterophile antibody to which the cell lines are particularly sensitive. Nonspecific lysis of target cells by SDC in the antigen fractions was reduced by diluting the fractions in RPMI 1640 medium containing 20 % (v/v) inactivated fetal calf serum.

3. Results 3.1. Cell surface location

Subcellular fractionation of homogenates of BRI 8 lymphoblastoid cells showed previously that HLA-A and B antigens are located preferentially, if not exclusively, o n the plasma (cell surface) membrane [ 181. Similar studies have now revealed that HLA-C antigens have an identical subcellular distribution with the HLA-A and B antigens. Thus, for example, the purified plasma membrane fraction of BRI 8 cells contained 42, 34 and 38 % of the HLA-A2, B8 and CW2 antigenic activities respectively, of the initial cell homogenate. It was concluded that HLA-C antigens are also located almost exclusively on the plasma membrane.

3.2. Molecular size The molecular size of the HLA-CW2 antigen was estimated from measurements of its gel filtration and sedimentation behavior in SDC. A purified preparation of BRI 8 plasma membrane was used as the source of HLACW2 antigen. Fig. 1 shows that HLA-CW2 antigen was eluted from a column of Ultrogel AcA 34 as a single, essentially symmetrical peak whose position coincided with that for P2m antigenic activity and with those found previously for HLA-A, B and Ia antigens (e.g. see Fig. 2, ref. [24]). This elution pattern is in marked

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Eur. J. Immunol. 1977.8: 580-585

D. Snary, C.J. Barnstable, W.F. Bodmer and M.J. Crumpton

i

n

an S20,w value of 5.1 5. This value was identical with that estimated previously for HLA-A1 and A2 antigens [21], but clearly differed from that of HLA-B8 antigen determined a t the same time (Fig. 2), and that reported previously for HLA-B antigens (S20,w 4.55)[ 211. The Stoke's radii and S20,w values of the HLA-A, B and C antigens and the molecular weights calculated from these values are summarized in Table 2. The HLA-A and C antigens possessed the same apparent molecular weight of 88 000 compared with 78 000 for the HLA-B antigens. As discussed previously [ 2 I], these values include bound SDC. Table 2. Molecular size of HLA-A, B and C antigens in SDC

Figure 1. Gel filtration on a column of Ultrogel AcA 34 of BRI 8 plasma membrane solubilized in SDC. The column was eluted with 0.5 % S I X 10 mM Tris-HCI buffer, pH 8.2, and the eluate was monitored for protein (0, E;;Znrn), HLA-CW2 activity (A)and &m (m). HLA-A, B and la activities were coincident with those shown for HLA-CW2 and &m. The apparent asymmetry of the antigen peaks is of questionable significance. Results obtained using various antiserum dilutions indicated that it was related to the sensitivity of the assay and suggested that it may represent nonspecific activity rather than aggregation.

contrast to the multiple peaks reported previously for the HLA-A, B and C antigens of a crude membrane fraction from human spleen that had been solubilized in Nonidet P40 [ 251. The basis of this difference has not been unequivocally defined, but it appears likely that it is related t o the use of crude membrane and is due to two factors, namely degradation [ 251 and disulfide interchange [ 2 11. Sucrose density gradient centrifugation of SDC-solubilized BRI 8 plasma membrane (Fig. 2) gave a single peak of HLA-CW2 activity with

Figure 2. Sucrose density gradient centrifugation of BRI 8 plasma membrane solubilized in SDC. Fractions were collected from the bottom of the gradient and were monitored for protein (0, E,&&rn), HLA-CW2 activity (A)and HLA-B8 activity (0).a m occupied a coincident position with the HLA-B8 and CW2 antigens but was not detected in the position of free &m at the top of the gradient (not shown). The asymmetry of the leading edge of the HLA-B8 antigen peak is of doubtful significance (see legend of Fig. 1).

Antigenic activity Al, A2 B8, 8 1 3 CW2

Stoke's radius (nm) 4.4 4.4 4.4

Bound SDCa)

S ~ O , Mol. ~ wt. 5.15 4.55 5.15

88000 18000 88000

(g/g of protein)

0.6 0.4 0.6

a) Calculated from the difference between the mol. wt. determined for the SDC-solubilized antigens and those estimated by polyaaylamide gel electrophoresis in sodium dodecyl sulfate for the component polypeptide chains (one each of 43 000 and 12 000 moL wt.).

3.3. Association with &m Antigens associated with P2m were depleted from a sample of SDC-solubilized BRI 8 plasma membrane by passage through a column of antibody against S2m attached to Sepharose. The effluent was monitored for protein and P2m, HLA-A2, CW2 and la antigenic activities. The results shown in Fig. 3 indicate that the majority of the membrane protein and the HLA-Ia antigenic activity emerged together and very much earlier than the HLA-A2, and CW2 activities whose position corresponded t o saturation of the column by B2m.

Figure 3. Immunoadsorption of BRI 8 plasma membrane with antibody against &m A sample of SDC-solubilized plasma membrane w a s eluted from a column of the lg fraction of an anti-&m serum attached to Sepharose 4B. The eluate was monitored for protein ( 0 , and HLA-A2 (A), HLA-CW2 (A), HLA-la (O), and &m (m) antgenic activities Although the elution of Pzm apparently preceded that of the HLA-A, B and C antigens, this difference was related to the sensitivity of the assay and was not significant.

Ei;Fn,rn),

Structure of HLA-C antigens

Eur. J. Immunol. 1977.8: 580-585

The retarded position of HLA-CW2 activity clearly indicates that the HLA-CW2 antigen resembles t h e HLA-A and B antigens in containing &m, whereas the lack of retardation of the HLA-Ia antigen endorsed the previous conclusion that this antigen is n o t associated with Pzm [24].

583

binds, or the addition of further sugar residues t o the carbohydrate chains that mask the N-acetylglucosamine/mannose units (c.J Thy-1 antigen from rat thymocytes and brain [ 261). The absence of a discernible peak of &m associated with the first peak of HLA-CW2 activity most probably reflects the low level of HLA-CW2 antigen (see below).

3.4. Association with carbohydrate

The results of various studies have shown that L. culinaris lectin binds specifically most lymphocyte plasma membrane glycoproteins including the majority of the HLA-A, B and la antigens [ 18, 22, 241. Indeed, adsorption t o lentil lectin and subsequent elution with the specific sugar has been employed as a diagnostic criterion of association with carbohydrate. The affinity chromatography of SDC-solubilized BRI 8 plasma membrane o n a column of lentil lectin-Sepharose is illustrated in Fig. 4 . The results showed that 12 % o f the membrane protein was bound and eluted with methylaD-mannopyranoside. The eluted glycoprotein fraction contained 86 % of the HLA-A2 activity but only 50 % of the HLA-CW2 activity. The remaining 14 % of the HLA-A2 activity was eluted together with the unretarded protein peak, whereas, in contrast, the other 50 % of the HLA-CW2 antigen emerged from the column just subsequent t o the unretarded protein, The latter result suggests that about half of the HLACW2 antigen had a much lower affinity for lentil lectin than the remainder and that the less avid portion underwent successive adsorption and elution. It was concluded that the HLACW2 antigen resembles the HLA-A and B antigens in being glycosylated, but that its carbohydrate moiety is heterogeneous. It has yet t o be determined whether t h e lower affinity for lentil lectin indicates incomplete addition of N-acetylglucosamine and/or mannose residues, to which the lectin

1”

‘ I

c

I

II

Figure 4. Affinity chromatography of BRI 8 plasma membrane on Lens culinuris lectin-Sepharose. A sample of SDC-solubiIized plas-

ma membrane was eluted from a column of L. culinrvis lectinSepharose with 0.5 % SDC 10 mM Tris-HCl buffer, pH 8.2. At frae tion no. 38 the adsorbed glycoprotems were eluted by washing the column with 2 % methyl-cI-Dmannopyranoside in 0.5 % SDC 10 mM Tris-HCI buffer, pH 8.2. The eluate was monitored for protein ( (0, Ei;rn,,,) and HLA-A2 (A), HLA-CW2 (A)and pzm (m) antigenic activities

3.5. Relative content of HLA-A,

B and C antigens

Estimation of the relative amounts of HLA-A, B and C antigens o n BRI 8 cells from inhibition data is not possible, since different antisera, even of the same apparent specificity, require different amounts of antigen t o cause a 50 % reduction in cytotoxic titer. This difference probably reflects variation in the relative amounts of different antibody types (IgM and/ or IgG), their affinities and specificities. A comparison of the distributions of HLA-A, B and C, and &m activities on lentil 1ectinBepharose (Fig. 4) suggests, however, that HLA-CW2 antigen is expressed t o a much lower extent (< 10 %) than the HLA-A and B antigens. This suggestion is based on the observation that all of the HLACW2 activity is apparently associated with Pzm (Fig. 3), and o n the assumption that all of the &m of the glycoprotein fraction eluted from lentil lectin-Sepharose is accounted for by the HLA-A, B and C antigens. In this case, the level of PZm activity underlying the first peak of HLACW2 activity in Fig. 4 reflects the amount of HLACW2 antigen and may be used t o calculate the amount of HLACW2 antigen relative t o t h e HLA-A and B antigens in the glycoprotein fraction. 3.6. Distribution of Pzm During the above experiments the following information was collected o n the distribution of Pzm. The &m activities of the various subcellular fractions obtained during fractionation of the BRI 8 cell homogenate paralleled the content of HLA-A, B and C antigens. N o pool of free Pzm was detected in the cytosol fraction. Free &m was also not detected in the SDC-sohbilized plasma membrane fraction by either gel filtration or sucrose density gradient centrifugation (Figs. 1 and 2). The latter results have an added significance since various studies suggest that free P2m was much more readily detected than an equivalent amount of the bound protein. Thus, a comparison of the capacities of free (human urinary) and bound (BRI 8 plasma membrane) &m t o inhibit the rabbit anti-&m serum suggested that the free protein was a more effective inhibitor (about 100-fold) than the bound polypeptide. (The amount of bound Pzm added was based o n the assumption that it was all associated with the HLA-A, B and C antigens which collectively represented about 1 % of the membrane protein). This result is consistent with the observation that heating BRI 8 plasma membrane at 40 OC for 15 min had a minimal effect on the HLA-A, B and Ia activities, but increased the &m activity 3-fold (Fig. 5), as would be expected if some of the bound Pzm had been dissociated. These results argue in support of the view that all of the &m of BRI 8 cells is bound t o the polymorphic chains of the HLA-A, B and C antigens. In contrast, the cytosol of human placenta [27] and spleen (unpublished results) apparently contained some free &m. I t is not yet clear whether this variation reflects a difference in P2m metabolism in different cell types, or whether the lack of free Pzm is peculiar t o BRI 8 cells. Recent reports that a lymphoblastoid cell line and a crude membrane fraction

584

Eur. J. Immunol. 1977.8: 580-585

D. Snary, C.J.Barnstable, W.F. Bodmer and M.J. aumpton

from human spleen have free &m [25, 281 should be viewed with caution since, as the authors pointed o u t , the techniques used may well have promoted dissociation of the bound polypeptide.

Figure 5. Effect of heating BRI 8 plasma membrane on antigenicity. The HLA-A2 (A), HLA-la (0)and &rn (m) antigenic activities were determined on samples of BRI 8 plasma membrane that had been

heated for 15 min at various temperatures a m was assayed using the rabbit antiserum donated by Dr. Grey, and HLA-A2 activity was measured using serum F3002X The HLA-la activity represents the average of results obtained using various allogeneic and xenogeneic antisera [20]. The results are expressed relative to the activity of a control sample of membrane that had been stored at 0 OC. Note that membrane which had been heated at 40 OC showed a >fold increase in &m activity. 4. Discussion

The current results indicate that the products of the H L A C locus are structurally homologous t o the HLA-A and B gene products. A similar interpretation was derived recently using similar approaches t o those described above [ 251 and, alternatively, by comparing maps of the tyrosine-containing peptides of immune precipitated 1251-labeled HLA-A, B and C antigens [ 291. The latter results are, however, difficult t o interpret for various reasons, including the presence of shared peptides derived from the common &m component, t h e poor resolution of one-dimensional separation techniques and the coprecipitation of 1z51-labeled actin by indirect immunoprecipitation of cells that have been labeled by lactoperoxidasecatalyzed iodination (B.H. Barber, M.J. Crumpton, D. Snary and F.S. Walsh, unpublished observation). In the present study, the homologous structure of the HLA-A, B and C antigens was established by the following studies. Immunoadsorption with anti-p2m clearly showed that essentially all of the HLA-A, B and C antigenic activities of SDC-solubilized BRI 8 plasma membrane were associated with Pzm, since no alloantigenic activity was detected prior t o saturation of the adsorbent with &m (Fig. 3). The immunoadsorbent also bound polypeptides of 1 2 000 (&m) and 4 3 000 molecular weight only from a glycoprotein fraction of BRI 8 plasma membrane containing HLA-A, B, C and Ia antigens [30]. Although only about half of t h e H L A C antigen was adsorbed by lentil lectin and specifically eluted with sugar compared with more than 8 5 % o f t h e HLA-A and B antigens, the remaining H L A C antigen was significantly retarded

relative t o t h e nongfycosylated protein (Fig. 4). As a result, the variation in HLA-C antigen behavior most probably reflects heterogeneity of carbohydrate structure (c.f. rat thymocyte Thy-I antigen [ 2 6 ] ) ,in which case all of the H L A C antigen is glycosylated. The HLA-A, B and C antigens behaved in an identical manner o n gel filtration, but the HLA-A and C antigens sedimented at a faster rate than the HLA-B antigens o n sucrose density gradient centrifugation (Table 2). The apparent closer structural similarity of H L A C antigen t o HLA-A than HLA-B antigen in SDC is interesting, since it appears t o be contrary t o expectation from genetic recombination studies which have placed the C locus closer t o the B than the A locus [ S ] . In this connection it will be of interest t o determine whether the amino acid sequence of the H L A C polypeptide also resembles more closely that of the HLA-A than t h e HLA-B antigen. The difference between the molecular weight of the SDC-solubilized H L A C antigen (88 000) calculated from gel filtration and sedimentation studies, and the sum of the molecular weights of the polypeptide chains ( 5 5 000) most probably represents SDC bound t o the nonpolar portion of the polymorphic chain that dips into the membrane lipid bilayer [21]. In this case, the variation in sedimention behavior of the SDC-solubilized H L A C and B antigens reflects different amounts of bound detergent and suggests that the H L A C 43 000 molecular weight chain is intercalated more extensively into the lipid bilayer than the HLA-B polypeptide. Although the present work is based exclusively upon the analysis of the HLACW2 antigen of BRI 8 lymphoblastoid cells, similar interpretations have been derived for the HLA-CW3 antigen from human spleen (251. As a result, it appears likely that the overall conclusion is independent of the source and specificity of the H L A C antigen. Similarly, the HLA-A and B antigens of various lymphoblastoid celllines and tissues behave in an identical manner with those of BRI 8 cells. It has not proved possible t o determine the relative amounts of HLA-A, B and C antigens o n BRI 8 cells, although t h e results shown in Fig. 4 indicate the relative paucity of the H L A C antigen. A lower level of HLA-C antigen may account for the problems experienced in defining the serological specificities o f the C locus since the antigens would be more difficult t o detect. Another explanation for the poor definition of H L A C antigens is that it is difficult t o obtain antisera devoid of contaminating HLA-B antibodies. Since it has been suggested that the polymorphism of the HLA-A, B and C antigens may represent differential expression of one of a cluster of structuralgenes [31], it is also possible that the high blank frequency for t h e HLA-C locus may actually represent true blanks in which no HLA-C antigen is expressed. The structural relationships that are ultimately established between the products of the HLA-A, B and C loci will be of value in elucidating the evolution of these loci from a presumptive precursor gene(s). It remains t o be determined whether the small differences in molecular parameters between the HLA-A, B and C gene products that have been revealed during this study are correlated with differences in biological function. We express our gratitude to Julicr G. Bodmer for the HLA-A, B, C and Ia typing sera, to Howard Grey for the rabbit anrCp2rn serum and to PeterJ. Lachman for the human urinary pzm CLB. ocknow ledges the receipt of a MRC Research Studentship.

Received May 3,1977.

Human MIF tested on mouse bone marrow macrophages

Eur. J. Immunol. 1977. 8 585-588 5. References 1 Bodmer, J., in Kissmeyer-Nielsen, F. (Ed.), Histocompatibility Testing 1975, Munksgaard, Copenhagen 1975,p. 21. 2 Moller, C. (Ed.), Transplant. Rev. 1974.vol. 21. 3 Sandberg, L., Thorsby, E., Kissmeyer-Nielsen, F. and Lindholm, A., in Terasaki, P.I. (Ed.), Histocompatibility Testing 1970, Munksgaard, Copenhagen 1970,p. 165. 4 Svejgaard, A., Staub Nielsen, L, Ryder, L.P., Kissmeyer-Nielsen, F., Sandberg, L., Lindholm, A. and Thorsby, E., in Dausset, J. and Colombani, J. (Eds), Histocompatibility Testing 1972, Munksgaard, Copenhagen 1973,p. 465. 5 Bodmer, W.F., Cytogenet. Cell Genet. 1976. 16: 24. 6 Cresswell, P., Turner, M.J. and Strominger, J.L, Proc. Nut. Acad Sci US 1973. 70: 1603. 7 Tanigaki, N., Katagii, M., Nakamuro, K, Kreiter, V.P. and Pressman, D., Immunology 1974. 26: 155. 8 Bridgcn, J., Snary, D., Crumpton, M.J., Barnstable, C., Goodfellow, P. and Bodmer, W.F., Nature 1976.261: 200. 9 Terhorst, C., Parham, P., Mann, D.L. and Strominger, J.L, Roc. Nut. Acad. Sci. US 1976. 73: 910. 10 Appella, E., Tanigaki, N., Fairwell, T.and Pressman, D., Biochem Bwphys Res Commun. 1976. 71: 286. 1 1 Ballou, B., McKean, D.J., Freedlender, E.F. and Smithies, 0.. Proc. Nut. Acad. Sci US 1976. 73: 4487. 12 van Someren, H.,. Westerveld, A., Hagemeijer, A., Mees, J.R., Meera Khan, P. and Zaalberg, O.B., Proc. Nut. Acad. Sci US 1974. 71: 962. 13 Jones, E.A., Goodfellow, P.N., Kennett, R.H. and Bodmer, W.F., Somat. Cell Genet. 1976.2: 483. 14 Goodfellow, P.N., Jones, E.A., van Heynhgen, V., Solomon, E., Bobrow, M., Miggiano, V. and Bodmer, W.F., Nature 1975.254~267 15 Arce-Gomez, B., Jones, E.A., Barnstable, C.J., Solomon, E. and Bodmer, W.F., Tissue Antigens 1977,in press.

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16 Poulik, M.D., Bernoco, M., Bernoco, D. and Ceppellini, R., Science 1973. 182: 1352. 17 Mayr, W.R., Bernoco, D., de Marchi, M. and Ceppellini, R., nansplant. Roc 1973.5 : 1581. 18 Snary, D., Goodfellow, P., Hayman, M.J., Bodmer, W.F. and Crumpton, M.J., Nature 1974.247: 457. 19 Dausset, J. and Colombani, J. (Eds.), Histocompatibility Testing 1972, Munksgaard, Copenhagen 1973. 20 Barnstable, C.J., Jones, E.A., Bodmer, W.F., Bodmer, J.C., ArceC o m a , B., Snary, D. and Crumpton, M. J., Cold Spring Harbor Symp. Quant. Biol 1917.41:443.

21 Snary, D., Goodfellow, P., Bodmer, W.F. and Chumpton, M.J., Nature 1975.258: 240. 22 Hayman, M.J. and Crumpton, M.J., Biochem Biophys R e s Commun. 1972.47 923. 23 Bodmer, W.F., in Ray, J.G., Hare, D.B. and Kayhoe, D.E. (Eds.), Manual of Tissue Typing Techniques, DHEW PubL no. (NIHNIAID) 74-5451973,p. 18. 24 Snary, D., Barnstable, C.J., Bodmer, W.F., Goodfellow, P.N. and Crumpton, M.J., Scand J. Immunol 1977.6 : 439. 25 Rask, L., Lindblom, J.B. and Peterson, P.A., Eur. J. lmmunol. 1976. 6: 93. 26 Barclay, A.N., Letarte-Muirhead, M., Williams, A.F. and Faulkes, RA., Nature 1976.263: 563. 27 Goodfellow, P.N., Barnstable, C.J., Bodmer, W.F., Snary, D. and Crumpton, M.J., Transplantation 1976. 22: 595. 28 Nakamuro, K., Tanigaki, N. and Pressman, D., Immunology 1977. 32: 139. 29 Cunningham-Rundles, C., Jersild, C., Svejgaard, A., Dupont, B. and Good, R.A., h c . Nut. Acad Sci US 1975. 72: 5081. 30 Snary, D., Goodfellow, P., Bodmer, W.F. and Crumpton, M.J., in Acton, R.T. (Ed.), Roc Cell Cblture Con$, Birmingham, A l a , Academic Press, New York 1977. 31 Bodmer, W.F., Transplant Proc. 1973.5 : 1471.

Short Papers Marie-Luis Lohmann-Matthes, W. Domzig and H.-G. Meerpohl Max-Planck-lnstitutfur Immunbologie, Freiburg/Brsg.

Mouse bone marrow-cultured macrophage as indicator cells for mouse and human migration inhibitory factor (MI F) Macrophages cultured from mouse bone marrow served as excellent indicator cells for mouse as well as for human migration inhibitory factor (MIF) preparations. The tests were performed in the capillary system and showed high reproducibility. Using mouse or human MIF-containing supernatants, we found an inhibition of 53 % compared with the controls. Similar results were obtained if concanavalin A-stimulated human peripheral blood lymphocytes were mixed with mouse test macrophages in a ratio of 1 : 5 or 1 : 10.

1. Introduction

The macrophage migration inhibitory factor (MIF) is one of the most frequently tested lymphocyte mediators [ 11. The MIF test is used t o detect delayed-type hypersensitivity in

[I 17081 Correspondence: Marie-Luise Lohmann-Matthes, Max-Planck-lnstitut

f* Immunbiologie, D7800 FreiburglBrsg., Postfach 1169,FRG Abbreviations: MIF: Migration inhibitory factor PCS: Fetal calf serum DMSO: Dimethylsulfoxide Con A Concanavalin A

research as well as in clinical applications. To be effective, a biological assay must be reproducible when carried out under standardized conditions. For the MIF test, however, the availability of suitable macrophages is a major problem for its routine application. Usually either guinea pig or mouse peritoneal macrophages elicited by peritoneal injection are used as test cells. In the case of guinea pig macrophages it is rather impractical t o kill one animal for a single test. On the other hand, the number of mouse peritoneal exudate cells obtained from a single animal is limited even when the exudates are induced with thioglycollate. The main disadvantage of these conventional sources is more fundamental. It is common ex-

Molecular structure of human histocompatibility antigens: the HLA-C series.

580 D.Snary, C.J. Barnstable, W.F. Bodmer and M.J. Oumpton Eur. J. ImmunoL 1977.8: 580-585 D. Snary", C.J. Barnstable', W.F. BOdmer' and M.J. Crump...
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