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Biochemical and molecular analysis of sheep MHC class II molecules
Veterinary Immunology and Immunopathology, 17 (1987) 231-242 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
231
BIOCHEMICAL AND MOLECULAR ANALYSIS OF SHEEPMHC CLASS I I MOLECULES N.K. PURl, P.C. SCOTT, C.L. CHOI and M.R. BRANDON Department of Veterinary Preclinical Sciences, The University of Melbourne, Parkville, Victoria 3052, Australia
ABSTRACT Puri, N.K., Scott, P.C., Choi, C.L., and Brandon, M.R., 1987. Biochemical and molecular analysis of sheep MHL class I I molecules. Vet. Immunol. Immunopathol. 17: 231-242. A panel of monoclonal antibodies was used for structural and immunodepletion analysis of sheep MHC class I I molecules. The results indicate the antibodies recognize molecules of molecular weight 32-34,000 (a chain) and 26-28,000 (B chain). Immunodepletionanalysis indicates that the antibodies may recognize up to four d i s t i n c t class I I molecules some of which are s t r u c t u r a l l y distinguishable using SDS-PAGE. Southern blot analysis using HLA-D region DR, DQ, DP, DO and DZ cDNA probes showed that a number of the cDNA probes hybridized specifically to sheep DNA indicating the presence of closely related genes in sheep. Together the results suggest that the sheep MHC class I I region contains distinct MHC class I I genes similar to those found in man. INTRODUCTION The Major Histocompatibility Complex (MHC) is a multigene family mediating a number of immunological functions (Sheffler and David, 1975; Klein, 1979; Klein et a l . , 1983).
Two groups of polymorphic cell surface glycoproteins coded for
by the MHC are the effector molecules intimately involved in immune recognition. One group, MHC class I molecules (44-48,000 dalton polypeptides associated with B 2 microglobulin) are the ubiquitously expressed classical transplantation antigens involved in graft rejection, and recognition of foreign antigens by T lymphocytes (Zinkernagel and Doherty, 1974), serving specifically as restriction elements for antigen recognition by cytotoxic (CD8 positive) T cells.
The
second group, MHC class I I molecules consist of two non-covalently associated subunits of 32-36,000 daltons (~chain) and 25-28,000 daltons (B chain) and are expressed predominantly on B lymphocytes and macrophages (Hammerling et a l . , 1976). Class I I molecules are involved in mixed lymphocyte reactions and graft versus host reactions as well as being involved in T cell-macrophage and T-B cell interactions as restricting elements for T helper (CD4 positive) cells. Extensive molecular analyses of the MHC of mouse and man have identified multiple class I I genes which are arranged as a number of closely linked gene clusters or l o c i .
In man there are up to five l o c i , DR, DQ, DO, DZ and DP and
in the mouse 2 l o c i , I-A and I-E. 0165-2427/87/$03.50
Individual loci contain genes for a number of
© 1987 Elsevier Science Publishers B.V.
232
distinct
~ and B molecules (Bell et a l . , 1985), each molecule consisting of two
extracellular domains ( e l , ~2 or B1, B2), a connecting peptide, a transmembrane region and a cytoplasmic t a i l . Until recently MHC-linked immune response genes or class I I molecules had not been identified in sheep (Chardon et a l . ,
1985; Puri et a l . ,
1985) although
serologically detectable sheep lymphocyte alloantigens had been described by a number of investigators (Ford, 1975; Cullen et a l . ,
1982; M i l l o t ,
1984).
However no information on the biochemical nature of these antigens or evidence for their linkage with genes controlling graft rejection or mixed lymphocyte reactions was available.
In this study the elucidation of the structural
characteristics and molecular organization of sheep MHC class I I
genes and
molecules was approached by producing a panel of MHC class
If-specific
monoclonal antibodies and by Southern blot analysis using Human MHC class I I gene probes. MATERIALS Monoclonal antibodies Monoclonal antibodies to sheep class I I molecules were produced using a number of immunogens including alveolar macrophages, efferent lymphocytes from cannulated lymph nodes and Lentil lectin purified lymphocyte glycoproteins.
An
additional monoclonal antibody was raised by an alloimmunization between two
TABLE I Monoclonal antibodies to sheep MHC class I I molecules
Monoclonal antibody* SBU.I I
Isotype
Immunogen
28-I
IgG1
Alveolar macrophages
37-68
IgG2a
A.TL splenocytes
38-64
IgG1
Efferent lymphocytes
38-27
IgG1
Efferent lymphocytes
38-30
IgG1
Efferent lymphocytes
42-20
IgG1
Lentil lectin purified glycoproteins
49.1
IgG2a
Efferent lymphocytes
*All monoclonal antibodies are monomorphic
233
congenic mouse strains (A.TH anti A.TL), with hybridoma supernatant subsequently selected for reactivity with sheep lymp~ocytes. Details of these monoclonal antibodies are given in Table 1 and Puri et al., (1985). Purification of lymphocyte glycoproteins Details are presented in Puri et al., (1985). Briefly, crude membrane pellets were prepared according to the method of Standring & Williams (1978) and solubilized using Renex-3O (Walker and Reisfield, 1982; Tanagaki & Toshi, 1982). Glycoproteins were isolated using a Lentil lectin Sepharose 4B column prepared according to the method of Hayman and Crumpton (1972) and Springer et al., (1977). Cell surface labelling The structure of the molecules recognized by the monoclonal antibodies in Table 1 was determined following lactoperoxidase catalysed cell surface iodination of sheep splenocytes. Labelling, immunoprecipitation, SDS-PAGE and autoradiography were carried out as described by Mackay et al., (1985). Immunoperoxidase staining flow cytometry analysis These techniques were as described by Mackay et al., (1985) and Puri et al., (1985). Immunodepletion experiments Purified lymphocyte glycoproteins (25ug) were iodinated using the chloramineT method (Williams et al., 1977). Seven individual samples (1.4 x 107 cpm/5Oul) were precleared by repeated immunoprecipitation using one of seven monoclonal antibodies (Table 1), until counts bound by each antibody were within 10% of background (non-specific) counts. The seven depleted lysates were then each subdivided into seven fractions and each fraction reacted with one of the other six monoclonal antibodies. Reactivity with a monoclonal antibody was determined by counting Protein-A Sepharose bound immune complexes in a gamma counter. The seventh fraction was reacted with the original antibody and served as the control to demonstrate that the molecules recognized by a particular antibody had been satisfactorily removed. Southern blot hybridization analysis Southern blot analysis was performed using standard techniques (Maniatis et al., 1982). Gene Screen Plus (Du Pont) was used according to the manufacturers directions and lOug of sheep genomic DNA, digested overnight with either Eco RI, Bam HI or Hind I l l restriction endonucleases, was used for blotting. A number of human MHC class II-specific cDNA probes were used for Southern analysis
234
including clone pDCHI (DQm ; Auffrey et a l . ,
1982), p I I - B-1 (DQ # ; Wiman et
a l . , 1982), pDRH2 (DRa ; Lee et a l . , 1982), DR- ~-2 (DR# ; Long et a l . , 1982), DR- ~-I0 (DR B; Kapper and Strominger, unpublished), 8bml (DZm ; Trowsdale and Kelly, 1985) and ~ 163 (DOE ; Tonnelle et a l . , 1985).
A Pst I fragment of the
cosmid clond LCII (designated 11-13) was used as the DP-B probe (Trowsdale et a l . , 1984). Further details including hybridization and washing conditions are reported in Scott et a l . , (1986). RESULTS Production of monoclonal antibodies against sheep class I I molecules A d i f f e r e n t i a l screening procedure using immunoperoxidase staining on frozen sections of thymus and mesenteric lymph node was used for selecting putative class I I - s p e c i f i c monoclonal antibodies. tissues is shown in Figure 1.
were i n t e n s e l y stained t o g e t h e r e x t r a f o l l i c u l a r T cell areas.
with
(1)
inter-digitating
cells
in
the
Thymic tissue stained with a representative MHC
class I I monoclonal antibody (Figure staining:
The staining pattern with these two
B cell f o l l i c l e s in the lymph nodes, Figure 1(a),
lb),
revealed two main patterns
of
intense confluent medullary staining and (2) a r e t i c u l a r
staining pattern in the cortex.
Specificity for B cells was confirmed using two
col our immunofluorescence to identify slg + c e l l s .
Figure 2 shows that the
majority of MHC class I I positive cells were slg + while only a small population of slg- cells were MHC class I I positive. Structure of sheep MHC class I I molecules The structure of the molecules recognized by the monoclonal antibodies was determined by immunoprecipitation and SDS-PAGE analysis of cell surface labelled splenocytes.
The results are presented in Figure 3. components of
immunoprecipitated,
each containing respectively a number of d i s t i n c t m- and B
- l i k e polypeptides.
32-34,000
In each case two major
molecular weight
and 26-28,000
There were also differences
daltons
were
in both the number and
electrophoretic mobility of the m and B polypeptides recognized by a given monoclonal antibody. three ~ - l i k e
For example, the molecule recognized by SBU.I I 38-27 has
polypeptides and an
~ chain complex distinguishable from that
recognized by SBU.I I 37-68 and 42-20. Immunodepletion analysis To distinguish between the products recognized by the monoclonal antibodies a number of strategies were employed including selective depletion of iodinated MHC class I I molecules using a single monoclonal antibody and then using the same fraction to assess r e a c t i v i t y with other monoclonal antibodies. This was used to analyse the i n t e r - r e l a t i o n s h i p between the various monoclonal
235
Figure 1.
Immunoperoxidase staining of paraffin embedded sections of (a) mesenteric lymph node and (b) thymus using class II monoclonal antibodies (x 160). The lymph node section shows staining of an isolated B cell f o l l i c l e (BF) within the cortex together with surrounding interfollicular and medullary regions. The section of thymus illustrates the staining pattern within the cortical (C) and medullary (M) regions.
236
(A)
(B)
LOG GREEN FLUORESCENCE Figure
'2. Two colour imnunofluorescence analysis of sheep PBL. Monoclonal antibody reactive cells were labelled with red fluorescence using biotinylated donkey anti-mouse Ig followed by PE coupled avidin. sIg+ lymphocytes were labelled with green fluorescence using FITC conjugated Donkey anti-sheep Ig. Results are shown as contour plots, each contour enclosing differing numerical levels of fluorochrome labelled cells. (A) PBS control, (B) SBU.11 28-1 (red fluorescence) versus anti-Ig (green fluorescence). Fluorescence intensities are on a log scale.
Figure
3.
SDS-PAGE analysis of MHC class II molecules immunoprecipitated from Standard I4C I25I lactoperoxidase labelled sheep splenocytes. labelled molecular weight markers are indicated. Lane (a) Monoclonal antibody SBlJ.II 28-1, (b) 37-68, (c) 38-64, (d) 38-27, (e) 38-30, (f) 42-20, (g) 49-1, (h) negative control. Samples were non-reduced and run on a 12% polyacrylamide gel.
237 TABLE 2 Summary of i mmunodepletion analysis of purified, iodinated sheep lymphocyte membrane glycoproteins by putative sheep MHC class I I - s p e c i f i c antisera.
Monoclonal antibody u s e d for immunodepletion
M o n o c l o n aantibodies l used for secondary incubation*
28-1
37-68
28-1
-
37-68 38-27
+ +
+
42-20
÷
+
30-30/38-64/49.1
28-27
+
4 2 - 2 0 38-30/38-64/49.1
+
+
+
+
+ +
÷ +
÷
-
+
-
*Antibodies were used following extensive immunodepletion.
antibodies.
The results are summarized in Table 2.
To demonstrate that all the
molecules recognized by a given monoclonal antibody were removed prior to incubation of labelled glycoproteins with another antibody, the number of counts bound by each antibody after six cycles of immunodepletion is presented in Figure 4.
I t is evident that in all cases, depletion with a given monoclonal
antibody was complete
since the number of counts bound was the same as
background due to non-specific binding.
Immunodepletion results (Table 2) are
presented as either positive (+) or negative (-) reactions and indicate that the monoclonal antibodies vary in terms of the number of class I I molecules they recognize.
The monoclonal antibodies SBU.II 28-1,
37-68, 38-27 and 42.20
recognize d i s t i n c t , mutually exclusive MHC class I I molecules.
Depletion of the
MHC class I I pool with one of these antibodies does not effect the r e a c t i v i t y of any of the other three monoclonal
antibodies.
This r e l a t i o n s h i p holds
irrespective of the order in which the primary depletion is carried out or the order in which an antibody is used for secondary immunoprecipitation. In contrast, SBU.I I 38-30, 38-64, and 49.1 recognize a number of different MHC class I I molecules including those recognized by the other four monoclonal antibodies.
Prior depletion using SBU.11 38-30, 28-1, 37-68, 38-27 or 42-20
only proportionately decreases the number of counts bound by these three monoclonal antibodies, while prior depletion using SBU.II 38-30, 38-64 or 49.1 removes all the class I I molecules reactive with either SBU.11 28-1, 37-68, 3827 or 42-20. The number of class I I molecules bound by a given monoclonal antibody is also reflected in the number of counts bound per immunodepletion cycle, as shown in
238
c p m (10 -s ) 49.1
•
111 1o. 9
i
*
i
1
2
3
i
a
,
4
5
6
N ° of d e p l e t i o n s Figure 4.
Immunodepletion analysis showing counts bound ([1251] class I I enriched glycoproteins) by a given monoclonal antibody per immu.nodepletion cycle. The dotted line represents non-specific background counts.
Figure 4, with SBU.11 38-30, 38-64 and 49.1 binding the highest counts per cycle. Southern hybridization analysis Southern blot analysis was performed using seven different HLA-D region cDNA probes covering the recognized HLA-D region l o c i , DR, DQ, DP, DO and DZ. Blots represent digestion of sheep genomic DNA using Eco RI, Bam HI and Hind I l l . Figure 5 shows an example of Southern blot analysis using DR ~- and ~-chain
239
(b)
DR-(I
(o)
-.-10 8.6-"
Fi gure 5.
W
Southern blot analysis using (a) the DR Bchain cDNA probe and (b) the DR ~ chain cDNA probe. From l e f t to right lOug of sheep DNA was digested with Eco RI, Bam HI, or Hind I l l and hybridized with the 32p labelled probes. Hybridization and wash conditions were as described in Scott et a l . , (1986). An Eco RI/Hind I l l digest of X phage DNA was used as a molecular size marker.
specific probes.
It
is apparent that the hybridization pattern using the B
chain probe, Figure 5(a), is quite complex and may contain up to five or six hybridizing bands depending on the enzyme used.
This level of complexity was
common when using B chain probes irrespective of which HLA-D region locus they corresponded to (results not shown). In contrast when using an a chain probe a much simpler pattern was obtained as shown in Figure 5(b).
Again this pattern
was common to the a chain probes used, irrespective of locus s p e c i f i c i t y . DISCUSSION Biochemical and molecular analysis has identified in both mouse and man a number of members of the MHC class I I gene family.
In man, using monoclonal
antibodies and gene cloning there is evidence for at least three d i s t i n c t groups
240
of class I I molecules (DR, DQ, DP) while genes for an additional two l o c i , DO and DZ have also been cloned (Tonnelle et a l . , 1985; Trowsdale and Kelly, 1985). Previous studies (Puri et a l . , the
cellular
and t i s s u e
1985) and the data presented here show that
distribution,
as well
as the
physicochemical
characteristics of sheep class I I molecules are very similar to those of man and mouse.
In addition the r e a c t i v i t y of a xenogenic I-E k specific monoclonal
antibody SBU.I I 37-68 with sheep class I I molecules provided evidence for the phylogenetic conservation of determinants between mouse I-E and sheep MHC class II molecules. The existence in sheep of d i s t i n c t class I I molecules was studied using immunodepletion analysis and Southern h y b r i d i z a t i o n .
Immunodepletion
experiments using monoclonal antibodies indicated that the antibodies recognize up to
four d i s t i n c t
class
II
molecules some of which are s t r u c t u r a l l y
d i s t i n g u i s h a b l e using immunoprecipitation
and SDS-PAGE. Although there are
problems associated with using heterologous DNA probes, hybridization between loci
i n c l u d i n g cross-
products (most evident using ~ chain probes), the
presence of unique strongly hybridizing bands for six of the probes, (DQ~ and #, DRm
and
~ , DO B.
and DZ a) suggests that the sheep has genes and loci
representative of those found in man. This has been confirmed by using these probes to isolate genomic clones (Scott et a l . ,
1986) and compares favourably
with the biochemical evidence for d i s t i n c t class II molecules. The data presented here and in Scott et a l . , (1986) provides evidence for the subdivision of the sheep MHC class I I region into a number of loci similar to those found in mouse and man.
The genes within these loci code for d i s t i n c t
class I I molecules which are distinguishable using monoclonal antibodies and biochemical analysis.
Studies are in progress to isolate these genes by genomic
cloning and to purify the glycoproteins identified by the monoclonal antibodies to sheep MHC class I I molecules. ACKNOWLEDGEMENTS We appreciate the valuable assistance of Mr. K. Snibson and Miss C. Kerr. Mr. N.K. Puri Corporation
is an Australian Meat and Livestock Research and Development
(AMLDRC) scholar.
This study was supported by a grant from the
AMLDRC. REFERENCES
Auffrey, C., Korman, A.J., Roux-Dousset, Magali, Bono, R. and Strominger, J.L., 1982. cDNA clone for the heavy chain of the human B cell alloantigeen DCI: Strong sequence homology to HLA-DR heavy chain. Proc. Natl. Acad. Sci., 79: 6337-6341. Bell, J . I . , Denny, D.W. and McDevitt, H.O., 1985. Structure and polymorphism of murine and human class I I major histocompatibility antigens. Immunol. Rev., 84: 50-71.
241
Chardon, P., Kirsenbaum, K., Cullen, P.R., G r e f f r o t i n , C., Auffrey, C., Strominger, J.L., Cohen, D. and Vaiman, M., 1985. Analysis of the sheep MHC using HLA class I, II and C4 cDNA probes. Immunogenetics, 22: 349-358. Cullen, P.R., Bunch, C., Brownlie, J. and Morris, P.J., 1982. Sheep lymphocyte antigens: A preliminary study. Animal blood groups and biochemical genetics, 13: 149-159. David, C., Mes, T., McCormick, J. and Shreffler, D., 1976. Expression of individual la specificities on T and B cells. J. Exp. Med., 143: 218-224. Ford, C . H . J . , 1975. Genetic studies of sheep leucocyte antigens, J. Immunogenetics, 2:; 31-40. Hammerling, G.T., Mauve, G., Goldberg, E. and McDevit, H.O., 1975. Tissue distribution of Ia antigens; la on spermatozoa, macrophages and epidermal cells. Immunogenetics, 1: 428-437. Hayman, M.J. and Crumpton, M.J., 1977. Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinans phytohaemagglutin. Biochem. Biophys. Res. Common., 47: 923-930. Klein, J., 1979. The major histocompatibility complex of the mouse. Science, 203: 516-521. Klein, J., Figueroa, F. and Nagy, Z.A., 1983. Genetics of the major histocompatibility complex: the final act. Ann. Rev. Immunol., 1: 119-142. Lee, J.S., Trowsdale, J. and Bodmer, W.F., 1982. cDNA clones coding for the heavy chain of human HLA-DR antigen. Proc. Natl. Acad. Sci., 79: 545-549. Long, E.O., Wake, C.T., Strubin, M., Gross, N., Acolla, R.S., Carrel, S. and Mack, B., 1982. Isolation of distinct cDNA clones encoding HLA-DRBchains by use of an expression assay. Proc. Natl. Acad. Sci., 79: 7465-7469. Mackay, C.R., Maddox, J.F., Gogolin-Ewens, K.J. and Brandon, M.R., 1985. Characterization of two sheep lymphocyte differentiation antigens SBU-T1 and SBU-T6. Immunology, 55: 729-737. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular cloning: A laboratory manual. Cold Springs Harbour laboratory, p. 383. Millot, P., 1984. The OLA major histocompatibility complex of sheep. Expl. Clin. Immunogenet., 1: 31-42. Puri, N.K., Mackay, C.R. and Brandon, M.R., 1985. Sheep lymphocyte antigens (OLA) II. Major histocompatibility complex class II molecules. Immunology, 56: 725-733. Sheffler, D.C. and David, C.S., 1975. The H-2 major histocompatibility complex and the la immune response region. Genetic v a r i a t i o n , function and organization, Adv. Immunol., 20: 125-195. Scott, P.C., Choi, C.L. and Brandon, M.R., 1986. The genetic organization of the MHC class II region in sheep. Immunogenetics (submitted). Springer, T.A., Mann, D.L., De Franco, A.L. and Strominger, J . L . , 1977. Detergent solublization, purification and separation of specificities of HLA antigens from a cultured human lymphoblastoid line RPMI 4265. J. Biol. Chem., 252: 4682-4693. Standring, R. and Williams, A.F., 1978. Glycoproteins and antigens of membranes prepared from rat thymocytes after lysis by Shearing or with the detergent tween-4O. Biochem. biophys. Acta., 588: 85-96. Tanigaki, N. and Toshi, R., 1982. Assessments of the specificity of human la alloantisera and alloantigens by the use of radio-iodinated human la antigens. Tissue Antigens, 20: 1-21. Tonnelle, C., De Mars, R. and Long, E.O., 1985. DO ~: a new Bchain gene in the HLA-D region with a distinct regulation of expression. EMBOJournal 4: No. 11, 2839-2847. Trowsdale, J., Kelly, A., Lee, J., Carson, S., Austin, P. and Travers, P., 1984. Linkage map of two HLA-SB B and two HLA SB ~-related genes: an intron in one of the SB~ genes contains a processed pseudogene. Cell, 38: 241-249. Trowsdale, J. and Kelly, A., 1985. The human HLA Class II ~chain gene DZa is distinct from genes in the DP, DQ and DR subregions. EMBOJournal, 4: 22312237.
231
BIOCHEMICAL AND MOLECULAR ANALYSIS OF SHEEPMHC CLASS I I MOLECULES N.K. PURl, P.C. SCOTT, C.L. CHOI and M.R. BRANDON Department of Veterinary Preclinical Sciences, The University of Melbourne, Parkville, Victoria 3052, Australia
ABSTRACT Puri, N.K., Scott, P.C., Choi, C.L., and Brandon, M.R., 1987. Biochemical and molecular analysis of sheep MHL class I I molecules. Vet. Immunol. Immunopathol. 17: 231-242. A panel of monoclonal antibodies was used for structural and immunodepletion analysis of sheep MHC class I I molecules. The results indicate the antibodies recognize molecules of molecular weight 32-34,000 (a chain) and 26-28,000 (B chain). Immunodepletionanalysis indicates that the antibodies may recognize up to four d i s t i n c t class I I molecules some of which are s t r u c t u r a l l y distinguishable using SDS-PAGE. Southern blot analysis using HLA-D region DR, DQ, DP, DO and DZ cDNA probes showed that a number of the cDNA probes hybridized specifically to sheep DNA indicating the presence of closely related genes in sheep. Together the results suggest that the sheep MHC class I I region contains distinct MHC class I I genes similar to those found in man. INTRODUCTION The Major Histocompatibility Complex (MHC) is a multigene family mediating a number of immunological functions (Sheffler and David, 1975; Klein, 1979; Klein et a l . , 1983).
Two groups of polymorphic cell surface glycoproteins coded for
by the MHC are the effector molecules intimately involved in immune recognition. One group, MHC class I molecules (44-48,000 dalton polypeptides associated with B 2 microglobulin) are the ubiquitously expressed classical transplantation antigens involved in graft rejection, and recognition of foreign antigens by T lymphocytes (Zinkernagel and Doherty, 1974), serving specifically as restriction elements for antigen recognition by cytotoxic (CD8 positive) T cells.
The
second group, MHC class I I molecules consist of two non-covalently associated subunits of 32-36,000 daltons (~chain) and 25-28,000 daltons (B chain) and are expressed predominantly on B lymphocytes and macrophages (Hammerling et a l . , 1976). Class I I molecules are involved in mixed lymphocyte reactions and graft versus host reactions as well as being involved in T cell-macrophage and T-B cell interactions as restricting elements for T helper (CD4 positive) cells. Extensive molecular analyses of the MHC of mouse and man have identified multiple class I I genes which are arranged as a number of closely linked gene clusters or l o c i .
In man there are up to five l o c i , DR, DQ, DO, DZ and DP and
in the mouse 2 l o c i , I-A and I-E. 0165-2427/87/$03.50
Individual loci contain genes for a number of
© 1987 Elsevier Science Publishers B.V.
232
distinct
~ and B molecules (Bell et a l . , 1985), each molecule consisting of two
extracellular domains ( e l , ~2 or B1, B2), a connecting peptide, a transmembrane region and a cytoplasmic t a i l . Until recently MHC-linked immune response genes or class I I molecules had not been identified in sheep (Chardon et a l . ,
1985; Puri et a l . ,
1985) although
serologically detectable sheep lymphocyte alloantigens had been described by a number of investigators (Ford, 1975; Cullen et a l . ,
1982; M i l l o t ,
1984).
However no information on the biochemical nature of these antigens or evidence for their linkage with genes controlling graft rejection or mixed lymphocyte reactions was available.
In this study the elucidation of the structural
characteristics and molecular organization of sheep MHC class I I
genes and
molecules was approached by producing a panel of MHC class
If-specific
monoclonal antibodies and by Southern blot analysis using Human MHC class I I gene probes. MATERIALS Monoclonal antibodies Monoclonal antibodies to sheep class I I molecules were produced using a number of immunogens including alveolar macrophages, efferent lymphocytes from cannulated lymph nodes and Lentil lectin purified lymphocyte glycoproteins.
An
additional monoclonal antibody was raised by an alloimmunization between two
TABLE I Monoclonal antibodies to sheep MHC class I I molecules
Monoclonal antibody* SBU.I I
Isotype
Immunogen
28-I
IgG1
Alveolar macrophages
37-68
IgG2a
A.TL splenocytes
38-64
IgG1
Efferent lymphocytes
38-27
IgG1
Efferent lymphocytes
38-30
IgG1
Efferent lymphocytes
42-20
IgG1
Lentil lectin purified glycoproteins
49.1
IgG2a
Efferent lymphocytes
*All monoclonal antibodies are monomorphic
233
congenic mouse strains (A.TH anti A.TL), with hybridoma supernatant subsequently selected for reactivity with sheep lymp~ocytes. Details of these monoclonal antibodies are given in Table 1 and Puri et al., (1985). Purification of lymphocyte glycoproteins Details are presented in Puri et al., (1985). Briefly, crude membrane pellets were prepared according to the method of Standring & Williams (1978) and solubilized using Renex-3O (Walker and Reisfield, 1982; Tanagaki & Toshi, 1982). Glycoproteins were isolated using a Lentil lectin Sepharose 4B column prepared according to the method of Hayman and Crumpton (1972) and Springer et al., (1977). Cell surface labelling The structure of the molecules recognized by the monoclonal antibodies in Table 1 was determined following lactoperoxidase catalysed cell surface iodination of sheep splenocytes. Labelling, immunoprecipitation, SDS-PAGE and autoradiography were carried out as described by Mackay et al., (1985). Immunoperoxidase staining flow cytometry analysis These techniques were as described by Mackay et al., (1985) and Puri et al., (1985). Immunodepletion experiments Purified lymphocyte glycoproteins (25ug) were iodinated using the chloramineT method (Williams et al., 1977). Seven individual samples (1.4 x 107 cpm/5Oul) were precleared by repeated immunoprecipitation using one of seven monoclonal antibodies (Table 1), until counts bound by each antibody were within 10% of background (non-specific) counts. The seven depleted lysates were then each subdivided into seven fractions and each fraction reacted with one of the other six monoclonal antibodies. Reactivity with a monoclonal antibody was determined by counting Protein-A Sepharose bound immune complexes in a gamma counter. The seventh fraction was reacted with the original antibody and served as the control to demonstrate that the molecules recognized by a particular antibody had been satisfactorily removed. Southern blot hybridization analysis Southern blot analysis was performed using standard techniques (Maniatis et al., 1982). Gene Screen Plus (Du Pont) was used according to the manufacturers directions and lOug of sheep genomic DNA, digested overnight with either Eco RI, Bam HI or Hind I l l restriction endonucleases, was used for blotting. A number of human MHC class II-specific cDNA probes were used for Southern analysis
234
including clone pDCHI (DQm ; Auffrey et a l . ,
1982), p I I - B-1 (DQ # ; Wiman et
a l . , 1982), pDRH2 (DRa ; Lee et a l . , 1982), DR- ~-2 (DR# ; Long et a l . , 1982), DR- ~-I0 (DR B; Kapper and Strominger, unpublished), 8bml (DZm ; Trowsdale and Kelly, 1985) and ~ 163 (DOE ; Tonnelle et a l . , 1985).
A Pst I fragment of the
cosmid clond LCII (designated 11-13) was used as the DP-B probe (Trowsdale et a l . , 1984). Further details including hybridization and washing conditions are reported in Scott et a l . , (1986). RESULTS Production of monoclonal antibodies against sheep class I I molecules A d i f f e r e n t i a l screening procedure using immunoperoxidase staining on frozen sections of thymus and mesenteric lymph node was used for selecting putative class I I - s p e c i f i c monoclonal antibodies. tissues is shown in Figure 1.
were i n t e n s e l y stained t o g e t h e r e x t r a f o l l i c u l a r T cell areas.
with
(1)
inter-digitating
cells
in
the
Thymic tissue stained with a representative MHC
class I I monoclonal antibody (Figure staining:
The staining pattern with these two
B cell f o l l i c l e s in the lymph nodes, Figure 1(a),
lb),
revealed two main patterns
of
intense confluent medullary staining and (2) a r e t i c u l a r
staining pattern in the cortex.
Specificity for B cells was confirmed using two
col our immunofluorescence to identify slg + c e l l s .
Figure 2 shows that the
majority of MHC class I I positive cells were slg + while only a small population of slg- cells were MHC class I I positive. Structure of sheep MHC class I I molecules The structure of the molecules recognized by the monoclonal antibodies was determined by immunoprecipitation and SDS-PAGE analysis of cell surface labelled splenocytes.
The results are presented in Figure 3. components of
immunoprecipitated,
each containing respectively a number of d i s t i n c t m- and B
- l i k e polypeptides.
32-34,000
In each case two major
molecular weight
and 26-28,000
There were also differences
daltons
were
in both the number and
electrophoretic mobility of the m and B polypeptides recognized by a given monoclonal antibody. three ~ - l i k e
For example, the molecule recognized by SBU.I I 38-27 has
polypeptides and an
~ chain complex distinguishable from that
recognized by SBU.I I 37-68 and 42-20. Immunodepletion analysis To distinguish between the products recognized by the monoclonal antibodies a number of strategies were employed including selective depletion of iodinated MHC class I I molecules using a single monoclonal antibody and then using the same fraction to assess r e a c t i v i t y with other monoclonal antibodies. This was used to analyse the i n t e r - r e l a t i o n s h i p between the various monoclonal
235
Figure 1.
Immunoperoxidase staining of paraffin embedded sections of (a) mesenteric lymph node and (b) thymus using class II monoclonal antibodies (x 160). The lymph node section shows staining of an isolated B cell f o l l i c l e (BF) within the cortex together with surrounding interfollicular and medullary regions. The section of thymus illustrates the staining pattern within the cortical (C) and medullary (M) regions.
236
(A)
(B)
LOG GREEN FLUORESCENCE Figure
'2. Two colour imnunofluorescence analysis of sheep PBL. Monoclonal antibody reactive cells were labelled with red fluorescence using biotinylated donkey anti-mouse Ig followed by PE coupled avidin. sIg+ lymphocytes were labelled with green fluorescence using FITC conjugated Donkey anti-sheep Ig. Results are shown as contour plots, each contour enclosing differing numerical levels of fluorochrome labelled cells. (A) PBS control, (B) SBU.11 28-1 (red fluorescence) versus anti-Ig (green fluorescence). Fluorescence intensities are on a log scale.
Figure
3.
SDS-PAGE analysis of MHC class II molecules immunoprecipitated from Standard I4C I25I lactoperoxidase labelled sheep splenocytes. labelled molecular weight markers are indicated. Lane (a) Monoclonal antibody SBlJ.II 28-1, (b) 37-68, (c) 38-64, (d) 38-27, (e) 38-30, (f) 42-20, (g) 49-1, (h) negative control. Samples were non-reduced and run on a 12% polyacrylamide gel.
237 TABLE 2 Summary of i mmunodepletion analysis of purified, iodinated sheep lymphocyte membrane glycoproteins by putative sheep MHC class I I - s p e c i f i c antisera.
Monoclonal antibody u s e d for immunodepletion
M o n o c l o n aantibodies l used for secondary incubation*
28-1
37-68
28-1
-
37-68 38-27
+ +
+
42-20
÷
+
30-30/38-64/49.1
28-27
+
4 2 - 2 0 38-30/38-64/49.1
+
+
+
+
+ +
÷ +
÷
-
+
-
*Antibodies were used following extensive immunodepletion.
antibodies.
The results are summarized in Table 2.
To demonstrate that all the
molecules recognized by a given monoclonal antibody were removed prior to incubation of labelled glycoproteins with another antibody, the number of counts bound by each antibody after six cycles of immunodepletion is presented in Figure 4.
I t is evident that in all cases, depletion with a given monoclonal
antibody was complete
since the number of counts bound was the same as
background due to non-specific binding.
Immunodepletion results (Table 2) are
presented as either positive (+) or negative (-) reactions and indicate that the monoclonal antibodies vary in terms of the number of class I I molecules they recognize.
The monoclonal antibodies SBU.II 28-1,
37-68, 38-27 and 42.20
recognize d i s t i n c t , mutually exclusive MHC class I I molecules.
Depletion of the
MHC class I I pool with one of these antibodies does not effect the r e a c t i v i t y of any of the other three monoclonal
antibodies.
This r e l a t i o n s h i p holds
irrespective of the order in which the primary depletion is carried out or the order in which an antibody is used for secondary immunoprecipitation. In contrast, SBU.I I 38-30, 38-64, and 49.1 recognize a number of different MHC class I I molecules including those recognized by the other four monoclonal antibodies.
Prior depletion using SBU.11 38-30, 28-1, 37-68, 38-27 or 42-20
only proportionately decreases the number of counts bound by these three monoclonal antibodies, while prior depletion using SBU.II 38-30, 38-64 or 49.1 removes all the class I I molecules reactive with either SBU.11 28-1, 37-68, 3827 or 42-20. The number of class I I molecules bound by a given monoclonal antibody is also reflected in the number of counts bound per immunodepletion cycle, as shown in
238
c p m (10 -s ) 49.1
•
111 1o. 9
i
*
i
1
2
3
i
a
,
4
5
6
N ° of d e p l e t i o n s Figure 4.
Immunodepletion analysis showing counts bound ([1251] class I I enriched glycoproteins) by a given monoclonal antibody per immu.nodepletion cycle. The dotted line represents non-specific background counts.
Figure 4, with SBU.11 38-30, 38-64 and 49.1 binding the highest counts per cycle. Southern hybridization analysis Southern blot analysis was performed using seven different HLA-D region cDNA probes covering the recognized HLA-D region l o c i , DR, DQ, DP, DO and DZ. Blots represent digestion of sheep genomic DNA using Eco RI, Bam HI and Hind I l l . Figure 5 shows an example of Southern blot analysis using DR ~- and ~-chain
239
(b)
DR-(I
(o)
-.-10 8.6-"
Fi gure 5.
W
Southern blot analysis using (a) the DR Bchain cDNA probe and (b) the DR ~ chain cDNA probe. From l e f t to right lOug of sheep DNA was digested with Eco RI, Bam HI, or Hind I l l and hybridized with the 32p labelled probes. Hybridization and wash conditions were as described in Scott et a l . , (1986). An Eco RI/Hind I l l digest of X phage DNA was used as a molecular size marker.
specific probes.
It
is apparent that the hybridization pattern using the B
chain probe, Figure 5(a), is quite complex and may contain up to five or six hybridizing bands depending on the enzyme used.
This level of complexity was
common when using B chain probes irrespective of which HLA-D region locus they corresponded to (results not shown). In contrast when using an a chain probe a much simpler pattern was obtained as shown in Figure 5(b).
Again this pattern
was common to the a chain probes used, irrespective of locus s p e c i f i c i t y . DISCUSSION Biochemical and molecular analysis has identified in both mouse and man a number of members of the MHC class I I gene family.
In man, using monoclonal
antibodies and gene cloning there is evidence for at least three d i s t i n c t groups
240
of class I I molecules (DR, DQ, DP) while genes for an additional two l o c i , DO and DZ have also been cloned (Tonnelle et a l . , 1985; Trowsdale and Kelly, 1985). Previous studies (Puri et a l . , the
cellular
and t i s s u e
1985) and the data presented here show that
distribution,
as well
as the
physicochemical
characteristics of sheep class I I molecules are very similar to those of man and mouse.
In addition the r e a c t i v i t y of a xenogenic I-E k specific monoclonal
antibody SBU.I I 37-68 with sheep class I I molecules provided evidence for the phylogenetic conservation of determinants between mouse I-E and sheep MHC class II molecules. The existence in sheep of d i s t i n c t class I I molecules was studied using immunodepletion analysis and Southern h y b r i d i z a t i o n .
Immunodepletion
experiments using monoclonal antibodies indicated that the antibodies recognize up to
four d i s t i n c t
class
II
molecules some of which are s t r u c t u r a l l y
d i s t i n g u i s h a b l e using immunoprecipitation
and SDS-PAGE. Although there are
problems associated with using heterologous DNA probes, hybridization between loci
i n c l u d i n g cross-
products (most evident using ~ chain probes), the
presence of unique strongly hybridizing bands for six of the probes, (DQ~ and #, DRm
and
~ , DO B.
and DZ a) suggests that the sheep has genes and loci
representative of those found in man. This has been confirmed by using these probes to isolate genomic clones (Scott et a l . ,
1986) and compares favourably
with the biochemical evidence for d i s t i n c t class II molecules. The data presented here and in Scott et a l . , (1986) provides evidence for the subdivision of the sheep MHC class I I region into a number of loci similar to those found in mouse and man.
The genes within these loci code for d i s t i n c t
class I I molecules which are distinguishable using monoclonal antibodies and biochemical analysis.
Studies are in progress to isolate these genes by genomic
cloning and to purify the glycoproteins identified by the monoclonal antibodies to sheep MHC class I I molecules. ACKNOWLEDGEMENTS We appreciate the valuable assistance of Mr. K. Snibson and Miss C. Kerr. Mr. N.K. Puri Corporation
is an Australian Meat and Livestock Research and Development
(AMLDRC) scholar.
This study was supported by a grant from the
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