Eur. J. Immunol. 1991. 21: 1793-1800
Ulrich Moebius., Gabriela Kober., Anne Lise GriscelW, Thierry Hercende and Stefan C. Meuer. Abteitung Angewandte Immunologie., Deutsches Krebsforschungszentrum, Heidelberg and DB artement de Biologie Celldaire~,Institut Gustave Roussy, Viejuif
Functionally distinct CD8 isoforms
1793
Expression of different 0 8 isoforms on distinct human lymphocyte subpopulations Human CD8+ lymphocyte subpopulationswere analyzed for their expression of CD8 a and CD8 p subunits. Investigations with uncloned peripheral blood lymphocytes as well as cloned human natural killer and Tcell subpopulations demonstrate that CD3- natural killer cells, Tcell receptor y/S, and CD4+CD8+ Tcell clones express exclusively CD8 a gene products. Structural analysis of CD8 molecules demonstrates that CD8 a+@-T lymphocytes surface express 75-kDa CD8 d a homodimers whereas CD8 d/3lymphocytes express concomittantly two CD8 isoforms of different molecular masses (67 kDa and 75 kDa, respectively). Peptide mapping of these latter two isoforms suggests that CD8 is expressed as da homodimers and a/fi heterodimers on CD8 cells. Importantly,we found that the two CD8 isoforms behave functionally different. Thus, in contrast to CD8 dp+/CD8 a/a+ T lymphocytes, cytolytic activity of CD8 a/P-/CD8 a/a+ T cell clones was not inhibited by anti-CD8monoclonal antibodies and the latter were not induced to proliferate following CD3/CD8 cross-linking.
1 Introduction
gene products can be expressed as homodimers on mouse Lcells. In contrast, surface expression of CD8 fi proteins The leukocyte differentiation antigen CD8 is expressed on required cotransfection of the CD8 a gene and was linked to various subpopulations of PBL: (a) a subset of the expression of CD8a molecules [20,21]. Homodimeric CD3+/'EcRdfi Tlymphocytes [l], (b) a subset of vs. heterodimeric configuration of human CD8 chains CD3+/'EcRy/6Tlymphocytes [2,3] and (c) a minor popu- expressed on L cells were distinguishable by means of one lation of CD3- NK cells [4]. CD8+/TcRd p Tlymphocytes particular anti-CD8 mAb, namely 2ST85H7, which does are known to recognize Ag in the context of MHC class I not react with CD8a transfectants [18, 19, 221. molecules and to exhibit cytotoxidsuppressor function [1,5]. In contrast, TcR y/S T lymphocytes were reported to CD8 was demonstrated to bind MHC class1 molecules predominantly exhibit non-MHC-restrictedcytotoxicactiv- directly [23,24]. Moreover, mAb directed at CD8 were ity [6,7], although some y/S Tcells were reported to reported to modulateT lymphocyte activation and effector recognize antigen in association with MHC class I mole- function either by inhibition or, alternatively, stimulation cules [8,9] as well as MHC class I-like molecules (CD1, Qa, depending on the experimental conditions employed TL; [lo-121). Finally, CD3- NK cells exhibit MHC- [25-281. CD8 has been reported, therefore, to be involved unrestricted cytolytic activity towards a number of primary in lymphocyte activation not only by mediating cell-to-cell tumors and tumor cell lines [4,13]. contact but also by transmitting functional signals. The recent finding that CD8 is intracellularly linked to the CD8 exists as a dimeric membrane glycoprotein composed tyrosine kinase p5dCkhas provided a rational basis for such of two disulfide-linkedsubunits.The isolated murine CD8 a a view [29,30]. and CD8 p subunits (Ly-2, Ly-3) have a molecular mass of 32kDa and 37kDa, respectively, and are encoded by In the present report we describe the characterization of separate genes.The human CD8 molecule was described as CD8 molecules expressed on TcR df3+Tlymphocytes, a dimer of two 34-kDa subunits [14]. Initially,only one CD8 TcRy/G+ Tcells and CD3- NK cells. Whereas TcRa/p+ molecule was identified in SDS-PAGE and since only the T cells expressboth CD8 a and CD8 f3 chains,TcRy/S T cells CD8 a gene [15,16] was known, human CD8 was believed and NK cells express only CD8 a as homodimners. Functo exist as a homodimer. After the identification of the tional analysis indicates that the C D 8 d a and CD8dp human CD8p gene [17], however, it became evident that forms, respectively, behave differently. Furthermore the both genes, CD8a and CD8p are expressed in CD8+ present data provide evidence that CD8 a and CD8 @ chains Tlymphocytes [18, 191 suggesting a heterodimeric s t r u ~ are coexpressed in two different dimeric isoforms on ture of this molecule. So far, CD8a and CD8p gene TcR a/@+Tcells. products have not been unequivocally defined at the protein level. 2 Materials and m e t W Transfection studies employing mouse and human CD8 a and CD8 (3 cDNA, respectively, have shown that the CD8 a
2.1 Cells and cell cnltnre conditions
Tcell clones were established from peripheral blood of healthy donors and from lymphocytic liver infiltrates of [I 94251 patients with chronic active B hepatitis, respectively. PBL were prepared by density centrifugation employing Ficoll* This work was supported by DFG-grant: Me 69314-1. Hypaque (Pharmacia, Freiburg, FRG). Liver-infiltrating thrreqmStefan C. Meuer, Abteilung Angewandte Immu- lymphocyteswere recovered as described [31]. Subsequentn w e , Iust. fiir Radiologie und Patkophysiologie, Deutsches ly, lymphocytes were stimulated with PHA (0.3 pg/ml, Kmhfmchungszentrum, Im Neuenheimer Feld 280, D-6900 Wellcome, Burgwedel, FRG} under limiting dilution conditons and individual clones were expanded as described Heidelberg, FRG 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+
OO14-2980/91/0808-1793$3.50 .2510
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U. Moebius, G. Kober, A. L. Griscelli et al.
[5,31]. PHA-activated T lymphocytes were generated by incubating PBL for 7 days in medium containing 0.3 pg/ml PHA.
Eur. J. Immunol. 1991. 21: 1793-1800
precipitation was performed for 4 h. Immunoprecipitates were washed, subjected to SDS-PAGE according to Laemmli [33] and visualized by autoradiography employing Kodak (Rochester, NY) X-Omat AR films.
2.2 mAb
CD3 mAb were OKT3 (purchased from American Type Culture Collection, Rockville, MD), RW28C8 (IgG1, kindly provided by Dr. E. Reinherz, Dana-Farber Cancer Institute, Boston, MA), and BMA030 (provided by R. Kurrle, Behringwerke, Marburg, FRG); CD4 mAb (12T4Dll) was provided by Dr. E. Reinherz. CD8 mAb were CD8.1 (AICD8.1, IgG1, fromour laboratory), CD8.2 (2ST85H7, IgGza), (338.3 (7PT3F9, IgG2,), CD8.4 (21Thy2D3, IgGI), all from Dr. E. Reinherz, CD8.5 (AICD8.2, IgG1) and CD8.6 (AICD8.3, IgG2,), both from our 1aboratory.TcR-specificmAb were BMA031 (IgG2b, R. Kurrle) and TcR61 (IgG1, TCell Sciences, Cambridge, MA). 2.3 Immunofluorescence analysis For indirect immunofluorescence,106 cells were incubated for 30 min with saturating concentrations of mAb (1 :1000 dilution of ascites fluid) in medium containing 5% human serum [31]. Subsequently,cells were washed and incubated with FITC-labeled goat-anti-mouse Ig Ab (Coulter, Hialeah, FL). For two-color immunofluorescenceanalysis, cells were incubated simultaneously with two different mAb followed by an incubation with a 1:40 dilution of two appropriate isotype-specific goat anti-mouse Ig Ab (Southem Biotechnology, Birmingham, AL) which were FITCand PE-labeled, respectively. Red (fluorescence 1) and green (fluorescence2) fluorescence was determined in a Coulter Epics Profile Cytofluorograph.
2.6 Northern blot analysis
RNA was extracted from 2 x 107-5 x lo7 Tcells according to the method of Chirgwin et al. [34]. Approximately 15 pg RNA was denatured employing 50% formamide, 18% formaldehyde in 1x Mops for 15 min at 55°C and was size-separated on 1% agarose gels employing 10% formaldehyde and 1x Mops as running buffer [35]. Subsequently, RNA was transferred to Gene Screen membranes (NEN, Dreieich, FRG) according to the manufacturer's instructions. About 100 ng of the specific gene probe was labeled by nick translation employing 750 kBq 32P-a-dCiT(NEN) and a nick translation kit (Boehringer Mannheim, Mannheim, FRG). Probes were a 1.3-kb EcoRI CD8a cDNA insert [15] and a 1.0-kb EcoRI CD8p cDNA insert [18], kindly provided by D. Littman, Stanford, CA, and a plasmid with inserted chicken actin cDNA. Filters were prehybridized in 50% formamide and hybridized overnight with 2 X lo6 cprdml labeled probe and 5 pg/ml salmon sperm DNA in prehybridization solution at 42°C. Filters were washed with 2 x SSC at room temperature (RT), 0.1 x S S C + l % SDS (65°C) and 0.1xSSC (RT) and exposed to Kodak XAR films for 1to 3 days. For a second hybridization with a different probe filters were washed in 100 m~ Tris, 10 mM EDTA, 1% SDS, pH 8.0, at 100°Cfor 20 min, prehybridized and hybridized as mentioned above. 2.7 Peptide mapping
After separation of immunoprecipitates, unfixed SDS gels were dried and exposed at -70 "C. Appropriate bands were excised. Peptide mapping employing Staphylococcus V8 CD3 mAb-induced cell mediated cytotoxicity was analyzed protease (Sigma) at 0.1 mg/ml was performed as described in standard Cr-release assays employing lo3 51Cr-labeled previously [36]. Daudi cells as targets [ 5 ] . Prior to addition of effector cells, labeled target cells were preincubated with CD3 mAb (OKT3) for 20 min. Tlymphocytes were preincubated in 2.8 Cell proliferation assay parallel with mAb CD8.5 (1 : lo00 dilution of ascites fluid) Wells of a 96-well flat-bottom plate (Nunc, Roskilde, and control mAb (14G3, same isotype as CD8.5), respec- Denmark) that were precoated overnight with a rabbit tively, for 20 min and added to target cells at an E/Tratio of anti-mouse Ig Ab (5 pg/ml, Dianova, Hamburg, FRG) 2/1. Following a 3-h incubation, released radioactivity was were incubated for 2 h with mAb BMA030 (300 ng/ml), or determined and % specific lysis was calculated according to CD8.4, or 12T4Dll (both 1: 10oO dilution of ascites fluid), the formula: or a combination of two mAb. Subsequently,7 x 104Tcells cpm - spontaneous release were added per well. After a 48-h incubation, cells were % Specific lysis = x loo maximum release - spontaneous release pulsed with 37 kBq [3H]dThd (NEN, 75 GBq/mmol) and harvested after an additional 24 h. 2.5 Immunoprecipitation and SDS-PAGE
2.4 Cytotoxicity assays
Cell surface labeling was performed as described previously [32]. Briefly, 4 x lo7 cells were labeled with 37 MBq lZI (816 MBq/pg, Amersham, Braunschweig, FRG) employing lactoperoxidase (Sigma, Munich, FRG) and glucose oxidase (Sigma). Cells were washed and lysed with 1% Triton X-100 (Sigma). After centrifugation at 10000 x g SN were subjected to immunoprecipitation. mAb used for immunoprecipitation were covalently coupled to protein A-Sepharose (Pharmacia) with dimethylpimelimidate (Sigma). BSA was added to cell lysates at 1mg/ml and lysates were precleared for 2 h with protein A-Sepharose (three times) followed by one additional preclearing step with rabbit anti-mouse Ig protein A-Sepharose. Specific
3 Results 3.1 Expression of Merent CD8 isoforms as defined by mAb
Recent cluster analysis of CD8 mAb employing CD8 transfectants expressing human CD8 a and CD8 p gene products showed that all CD8 mAb investigated,with the exception of one mAb (2ST85H7), recognized C D S d p heterodimeric as well as the CD8 d a homodimeric forms [22]. Since mAb 2ST85H7 was only reactive with Lcells co-transfected with CD8 a and CD8 p it was concluded that this mAb is specific for an epitope of the CD8 p chain or, alternatively, recognizes a composite epitope encoded by
Eur. J. Immunol. 1991.21: 1793-1800
Functionally distinct CD8 isoforms
both CD8 a and CD8 P. mAb 2ST85H7 is designated in the following as CD8.2. Additional mAb employed in this study which recognize CD8 cc in the absence of CD8 P are termed (338.1, CD8.3,CD8.4, (38.5 and CD8.6, respectively (see Sect. 2.2).
Table 1. Phenotype of human cloned lymphocytes Clones
"'1
0.31
10,9]
54
CD3
r
12dl
+ + + + + + + + + + + + + + +
5A2
To investigate whether the CD8 P-restricted epitope recognized by mAb CD8.2 is coexpressed with CD8a on all CD8+human PBL, two-color immunofluorescenceanalysis was performed. As shown in Fig. 1B for one out of four representative donors, a considerable number of CD8 a+ PBL (CD8.4+)were unreactive with mAb CD8.2 (7.5 out of 18.4%). Cells only reactive with mAb CD8.2 were not observed. Moreover, CD8.2 mAb reacts exclusively with CD3+T cells (Fig. 1D) whereas a fraction of GD3- T lymphocytes (NK cells) is stained with mAb CD8.3 (specificfor the CD8 a chain; Fig. 1C). Similarly, mAb CD8.2 is clearly unreactive with TcRCJP- Tcells (Fig. 1F) as well as TcRy/G+ cells (Fig. 1H). In contrast, surface expression of the CD8 a gene product (detectable with mAb CD8.3) is found in TcRa/P+ (Fig.lE), a fraction of TcRy/G+ cells (Fig. lG), and a proportion of CD3- cells (Fig. lC),
01
mAb OKT3 BMA031 TcRG CD4 CD8.1
JT9 C3M 3025 BMlA6 JT3 F5511CI F551IICI
SD7 7405 Bd Vtc
WT3166
WM2 WM3
remectivelv. It seems. t,.erefore. that exmession of 6 8 d P b; human Tlymphocytes is intimatiy linked to the presence of a TcR dp-CD3 complex. To investigate this point further, a series of cloned human CD8+ lymphocyte populations were analyzed with regard to their reactivity with mAb CD8.1 (a chain-specific) and mAb CD8.2, respectively. As shown in Table 1, all Tcell clones and CD3- NK cell clones listed were reactive with mAb CD8.1 (and mAb CD8.3-6, not shown). All CD8+ T cell clones expressing a TcR d p were reactive with mAb CD8.2. In marked contrast to TcRdP+ Tcell clones, TcR y/G+ T cell clones and CD3- NK clones were unreactive with mAb CD8.2. In addition to CD3- and the TcRy/G+
CD3
7 4 0 5 , (A) I r4 0)
LI
E 3 C 1
16
32
40
64
B M l A 6 , (A)
d
TcR-a6
4 0)
V
log fluorescence 1
-
.-
Figure 1. Itno-color immunofluorescence analysis. PBL were stained with pairs of mAb as indicated. Numbers inside the gated
areas of each histogram indicate the percentage of positive cells. Numbers on the histogram axis represent channel numbers of fluorescence.
log fluorescence Figure 2. Indirect immunofluorescence. Clones C3F2, 7405, BMlA6 and WM2 were stained with (A) negative control, (B) CD8.1 and (C) CD8.2, respectively. Fluorescence was determined on an Epics Profile Cytofluorograph by analyzing loo00 cells per sample.
Eur. J. Immunol. 1991. 21: 1793-1800
U. Moebius, G. Kober, A. L. Griscelli et al.
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clones, two TcRa/B Tcell clones were stained with mAb CD8.1 but not with mAb CD8.2 (WM2; WM3). Interestingly, these Tcell clones were found to stably coexpress CD4 and CD8 molecules as confirmed by means of two-color immunofluorescence (not shown). It should be noted that identical culture conditions were employed for all Tcell clones described here.
11684 58
The reactivity of four representative NK and T cell clones with mAb CD8.1 and CD8.2, respectively, is shown in Fig. 2. T cell clone C3F2 (BR a@) reacted with both types of CD8 mAb. In contrast, clones 7405 (TcRy/6), BMlA6 (NK), and WM2 ( R R d P , CD4+CD8+)were reactive with mAb CD8.1 but unreactive with mAb CD8.2. Expression of CD8 a by clones7405, BMlA6 and WM2 was lower than by CD8+I'IbRa@+ Tcell clones as judged from mean channel immunofluorescence (Fig. 2).
116
+
-.
48
36
84
--
58
-
48 +
36 26
-
Figure 4. CD8 immunoprecipitation. Immunoprecipitates from clones C3M (2),WM2 (3) and PBL (4) performed with negative control Ab (1) and CD8.1(2,3,4,5) were analyzed by SDS-PAGE on a 10% gel under nonreducing (A) and reducing (B) condi-
3.2 Expression of CDS a-and CD8 p-specific mRNA Representative Tcell and NK cell clones reactive with tions. either both types of CD8 mAb or CD8.1 only were subjected to Northern blot analysis to investigate the expression of CD8a and CD8P mRNA. As shown in o n n n o n Fig. 3, T cell clone C3F2 (lanes 1and 5 ) expressed CD8 a0 0 0 0 0 0 as well as CD8 P-specific mRNA. The TcR y/i3 Tcell clone 200 7405 (lane 2), the NK cell clone BMlA6 (lane 3) and T cell clone WM2 (lane 4) were found to express only CD8 a but not C D S P mRNA. A human B lymphoblastoid cell line 97 (M7) with no reactivity to any CD8 mAb did not express CD8 a and CD8 P genes (lane 6). 3.3 Immnnoprecipitation of individual CD8 isofonus
To characterize structurally the CD8 molecules of Tlymphocytes expressing either CD8a mRNA alone or along with CD8 p-specific mRNA, representative clones were surface labeled with lZsIand subjected to immunoprecipitation and SDS-PAGE. CD8.1 immunoprecipitates from Tcell clone C3F2 (CD8a@) and from PBL resulted in two 1
2
3
4
5
6
CD8-CI 18 s-
C D 8- A 18s
-
Actin 185
-
Figure 3. Northem blot andysis.Tital RNA of clones C3m (1, 5), 7405 (2), BMlA6 (3), WM2 (4), and the B-LCL M7 (6) was size-separated, transferred to a membrane and repeatedly hybridized with cDNA probes specific for CD8 a, CDS p and actin respectively.
68
-
43
-
29
-
Figure 5. CD8 immunoprecipitation. Immunoprecipitates from PHA-activated T lymphocytes employing various CD8 mAb were analyzed by SDS-PAGE on a 10% gel under nonreducing wnditions.
characteristic bands of 75 and 67 kDa when investigated under nonreducing conditions (Fig. 4A, lanes 2 and 4). In marked conrast, immunoprecipitates from clone WM2 (CD8da) resulted in only one band of 75 kDa (Fig. 4A, lane 3). Analysis of CD8 immunoprecipitates under reducing conditions showed a broad band of 31/32 kDa in the case of Tcell clone C3F2 and PBL (Fig. 4B, lanes 2 and 4). In contrast,WM2 gave a single and sharper band of 32 kDa (Fig. 4B, lane 3). To investigate which of these CD8 molecules is recognized by mAb CD8.2 and to compare several other CD8aspecific mAb we performed immunoprecipitatlon experiments*As shown in Fig- 5 9 mAb cD8-2 PreciPitated Only the 67-kDa molecule from lYsates of PHAactivated T lymphocytes. Besides mAb CD8.1, three additional CD8a mAb precipitated the 67 kDa molecule in
Functionally distinct CD8 isoforms
Eur. J. Immunol. 1991.21: 1793-1800
addition to the 75-kDa molecule (mAb CD8.4, CD8.5, CD8.6). In contrast mAb CD8.3 precipitated mainly the 75-kDa molecule (Fig. 5 ) . Note that mAb CD8.4, CD8.5 and CD8.6 precipitated different amounts of the two CD8 isoforms. 3.4 Peptide mapping of individual CD8 h f o m
The finding that GD8 d a Tlymphocytes express only the 75-kDa CD8 protein and that the 67-kDa molecule is related to an epitope recognized by the CD8P-restricted mAb CD8.2 suggests that the 75-kDa and 67-kDa proteins represent two CD8 isoforms of different subunit composition. To analyze further this question, we performed one-dimensional peptide maps. CD8 immunoprecipitates were separated by SDS-PAGE under nonreducing conditions, individual bands were excised and further subjected to V8 protease digestion and SDS-PAGE. As shown in Fig. 6, peptide mapping of 75-kDa proteins that were derived from CDSdP (C3F2) and C D 8 d a (WM2) T cell clones, respectively, resulted in identical fragments (lanes 2 and 3). Identity of peptide fragments was observed either when reducing (Fig. 6, lanes 2 and 3) or nonreducing conditions (Fig. 6, lanes 4 and 5) were employed for V8 protease digestion and SDS-PAGE.This suggests that the 75-kDa molecule expressed on CD8 dB T lymphocytes consists exclusively of CD8 a proteins and that this molecule can be considered as an a/a homodimer. In contrast, when the 75-kDa (Fig. 6, lane 2) and 67-kDa CD8 molecules (Fig. 6, lane 1) from a representative CD8 a/f3 T cell clone were compared, different peptide patterns were observed. Thus, digestion of the 67-kDa molecule resulted in one peptide (Fig. 6, position “c”) that was not present following digesiton of the 75-kDa molecule. In addition, fragments at position “d” were clearly distinct. Importantly, some fragments seem to be common 1
29
2
4
3
5
-a
14 -
-b
6-
-d
18
-C
3-
Figure 6. V8 protease peptide map of 75-kDa and 67-kDa CD8 molecules. CD8 immunoprecipitates from clones C3F2 (1, 2, 4) and WM2 (3,s) were subjected SDS-PAGE (10% gel) under nonreducing conditions. Subsequently, 75-kDa (2, 3, 4, 5) and 67-kDa (1)molecnles were excised, digested withV8 protease and analyzed on a 14% -20% gradient gel under reducing (1,2,3) and nonreducing (4,5) conditions.
200
-
97
-
68
-
43
-
1797
Figure 7. CD8 immunoprecipitation. Immunoprecipitates from Tcell clone 3025 performed with mAb CD8.2 and CD8.3, respectively,were analyzedby SDS-PAGE (10%) under nonreducing (A) and reducing (B) conditions.
for both proteins, particularly two fragments at position “a” and one at position‘%”. This suggests that the 67-kDa molecule in part consists of CD8a protein but contains additional protein(s). It should be noted that those fragments found to be common for both CD8 molecules were present with less relative intensity followingdigestion of the 67-kDa molecule.
To investigate further the subunit composition of the 67-kDa isoform we addressed the question of whether this dimer can be separated into two subunits. To this end, we employed mAb CD8.2 that precipitates only the 67-kDa molecule (Fig. 7A). For control purposes, the 75-kDa CD8 molecule was immunoprecipitated from a CD8+ NK clone employing mAb CD8.3. As shown in Fig. 7B, under reducing conditions the former precipitate yielded two separate bands of 32 kDa and 30 kDa, respectively. In contrast, reduction of the 75-kDa protein resulted only in the 32-kDa protein (Fig. 7B). Identical results were obtained when five additional CD8 dB T cell clones were analyzed (not shown). We then performed peptide map analysis of the 32 and 30-kDa polypeptides (compare Fig. 7B, CD8.2). As shown in Fig. 8, when the 32- and 30-kDa subunits were subjected separately to V8 protease treatment two widely differing peptide patterns were observed (Fig. 8, lanes 2 and 3). Thus, peptides at positions “a” and “b” were derived from the 32-kDa (Fig. 8, lane not from the 30-kDa molecule (Fig. 8, lane 3). resence of peptides at position “c” and “e” was less pronounced in the case of the 32-kDa than for the 30-kDa protein. This may result from cross-contamination due to incomplete separation of the subunits in SDS s at position “d” seem to represe in both, the 32and the 30-kDa molecule. However, the presence of two bands at this position was only observed for the 32-kDa
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U. Moebius, G. Kober, A. L.Griscelli et al. 1
2
Eur. J. Immunol. 1991.21: 1793-1800
Tmble 3. Prolilferative responses following CD3/CD8 stimula-
3
tion
clmr 17402
29 -
18
-
14 -
-a
Medium
-b
a33
-c
cm+cD8 m 3 cD8
W'
am
+
17411
4
a
144 116
us
76 54 142
6280
118 104 5492
5066
ND
ND
6s
a) mAb linked to tissue culture plate. b) rH]dThd uptake in cpm.
Figure 8. V8 protease peptide map of 32-kDa and 30-kDa CD8 molecules. CD8 immunoprecipitated from T cell clone 3025 employing mAb CD8.3 (1) and CD8.2 (2,3) were purified by SDS-PAGE on a 10% gel under reducing conditions. The 32-kDa (1,2) and 30-kDa (3) molecules were subsequently subjected toV8 protease treatment followed by SDS-PAGE on a 14%-20% gel under reducing conditions.
protein (Fig. 8, lanes 1 and 2). Importantly, peptide mapping of 32-kDa proteins, either derived from the 67-kDa dimer (lane 2) or the 75-kDa dimer (lane l), resulted in identical bands. Results analogous to those shown in Fig. 8 were also obtained when PHA-activated PBL were analyzed. These data support the view that the 67-kDa CD8 dimer consists of a 32-kDa CD8a subunit and a second 30-kDa subunit of different structure.
3.5 Functional analysis of CDS molecules expressed on CDSda and CDSdfl lymphocytes CD8 molecules were reported to be involved in T lymphocyte activation [25-28].Therefore,we were interested in the functional roles of the two different CD8 isoforms. To investigate this point, we studied the effect of CD8 mAb on the cytolytic activity of representative cloned lymphocytes (Table 2).To this end,various effector cell populations were pretreated with mAb prior to incubation in standard
Tmble 2. Functional effects of mAb on cytolytic activities of cloned effector cell populations
15D2
mAb 2lrfhp5D7
l2"4Dll
CD8.S
cKY8.4
cD4
50.8*1 22.3 28.9
9.2 ND 40.2
1.2 23.2 T.8
ND
12.2
12.0
Clone
Medium
Wr-release assays. As shown, the cytolytic activity of the CD4+/CD8a/a+ T cell clone WM2 towards its specific allogeneic target was not inhibited by mAb CD8.4. In addition, theTcR y/S T cell clone 7405 and the NK cell clone BMlA6 were not blocked by mAb CD8.5 andor CD8.4. In contrast, both CD8 mAb were strongly inhibitory for the Tcell clone C3F2. This inability of CD8 mAb to inhibit CD8 a/a lymphocytes could have been due to the fact that CD8 a/amolecules on these cells do not interact with MHC class I. Alternatively, functional effects independent of this CD8/MHC interaction could have been held responsible for this observation. To address the latter question,we employed Daudi cells, an FcR+, MHC class I- B lymphoma cell line which is not recognized by this set of Tcell clones. Addition of CD3 mAb (OKT3), however, brings together effector and target cells through binding to FcR and CD3, respectively, and triggers cytolytic effector function in a dose-dependent fashion. As shown in Fig. 9, CD3-induced cytotoxicity of Tcell clone C3F2 (CD8 a@+) was inhibited by addition of mAb CD8.5. In contrast, cytotoxicity of Tcell clone WM2 (CD8da) could not be blocked. This difference in CD8 mAb-mediated inhibition was observed at different levels of CD3-induced cytotoxicity. Note that inhibition of T cell clone C3F2 was specific for CD8 mAb since a control mAb of the same isotype did not result in reduction of cytolytic activity. Cross-linking of CD3 and CD8 (or CD3 and CD4) molecules was previously reported to induce proliferation of Okl-3
mAb
-
CD8
3x107 3x107 3x107 1x108 1x108 1x108
C3F2
CD8 Contr. ~
CD8 Contr
o
c3F2 wh42 BMlA6 1405
a) Percent specific lysis.
ND
WM2
10
10
30
u)
so
o
10
20
ao
.o
so
% SpeClfIC lpls
13.1
ND ND
Figure 9. Inhibition of cytotoxicity by CD8 mAb. C3F2 and WM2 cells, respectively,were incubated with 51Cr-labeledDaudi cells at an EfT ratio of 2/1. OK73 was added at 3 x and 10-8-fold dilution of ascites fluid. mAb CD8.5 was employed to study the function of CD8. Contr.: unrelated isotype-matched mAb.
Eur. J. Immunol. 1991. 21: 1793-1800
peripheral blood Tlymphocytes [25,26]. As shown in Table 3 when CD8 a / p Tcell clones were stimulated with submitogenic concentrations of surface-bound CD3 mAb, addition of CD8 a-specific mAb CD8.4 induced a proliferative response. In contrast, the CD4+/CD8a/a+/CD8a/pTcell clone WM2 was not induced to proliferate by CD3 plus CD8 mAb. This Tcell clone was, however, activated when CD4 molecules were cross-linked with CD3. These studies indicate that the two different CD8 isoforms expressed on C D 8 d a and C D 8 a / p Tlymphocytes serve different functions.
4 Discussion
Functionally distinct CD8 isoforms
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75-kDa disulfide-linkedhomodimers on the cell surface. In contrast, “conventional” CD8+ T lymphocytes were found to clearly express CD8 in at least two different dimeric isoforms, namely of 75 kDa and 67 kDa, respectively.The existence of two forms of the CD8 molecule (75 kDa and 67 kDa) was previously reported by others [14, 371. However, both molecules were believed to represent a/a homodimers with different intrachain disulfide bonds [38]. The present data demonstrate that the 75-kDa and 67-kDa molecules are composed of different subunits and that both isoforms can be co-expressed on unstimulated T cells and T cell clones.
Two separate genes were reported to encode for the CD8 a and the CD8 p subunits, respectively [15,17,20], and CD8 was known to be expressed as an a l p heterodimer on CD8+ Tlymphocytes [14, 37, 381. The present report demonstrates that CD8 is also expressed as an a/a homodimer on particular subsets of human lymphocytes. Thus, TcR y/S+ Tcells, CD3- NK cells, and RRa//P+, CD4+, CD8+ T cell clones were only reactive with CD8 a-specificmAb but did not bind mAb CD8.2 (2ST75H7) which reacts with CD8 p or a composite epitope of the a and P chains of CD8.Analysis of CD8 transcripts in a series of cloned human lymphocyte populations confirmed that they expressed CD8 a- but not CD8 P-specific mRNA consistent with the exclusive presence of CD8 a/a homodimers. In contrast, expression of CD8 p was only found on T lymphocytes expressing a TcR a@.
Following V8 protease treatment of the 67-kDa molecule those fragments common for the 75-kDa CD8 a/a molecule were relatively less intense as one would have expected assuming an a / p heterodimeric nature with 1: 1ratio of the two subunits. This might be explained with a different susceptibility to proteolysis of the CD8 a protein perhaps depending on the presence of the CD8 p molecule. Alternatively, it indicates that both proteins are not equally distributed and therefore not only present as heterodimers. It cannot be excluded that the 67-kDa immunoprecipitate consists of different CD8 dimeric isoforms which were coprecipitatedby mAb CD8.2.When under such conditions a/a homodimers were present, additional CD8 f3 protein would have been derived from fi/p homodimers. Since the exact specificity of mAb CD8.2 is not known, this, possibilitycannot be exclude at present. Note that this mAb is unreactive in Western blot analysis.
Cells expressing CD8 a/a homodimers represent minor subpopulations of human lymphocytes. Thus, a fraction of peripheral blood as well as tissue R R d p Tcells were reported to be CD8+ [2, 3, 39-41]. Similarly, a minor fraction of CD3- NK cells express CD8 [4]. Circulating T lymphocytes coexpressing CD4 and CD8 were only found at low frequencies in peripheral blood of normal donors [42]. In contrast, TcR a@ T lymphocytes expressing CD8 a and CD8p represent the majority of CD8+ peripheral lymphocytes [22,32]. These Tcells recognize antigen in combination with MHC class I molecules [5].
With regard to the specificityof various CD8 mAb our data indicate that those mAb precipitating both the 75-kDa and the 67-kDa molecules bind to epitopes of the a chain present in both isoforms. In contrast, mAb (338.2 and CD8.3 are specific for epitopes encoded by the a/p heterodimer and the a/a homodimer, respectively. Both CD8 isoforms could be noncovalentlyassociated on the cell surface and, therefore, coprecipitated. mAb CD8.2 and CD8.3, respectively, might disrupt the association of both isoforms because of their reactivity with epitopes that are crucial for CD8 ala-CD8 a l p interactions.
Transfection studies have previously demonstrated that the CD8 a protein can be expressed as an a/a homodimer on mouse L cells whereas the surface expression of the f3 chain required cotransfeciton of the a chain [20,21]. CD8 molecules consisting exclusively of fi chains have not been observed so far.
Previous findings that CD8+ Tlymphocytes were MHC class I restricted strongly suggested that CD8 binds directly to class I molecules. This was formally proven by binding studies employing reconstituted vesicles and transfected cells [23,24]. Since these transfection studies were performed employing CD8a genes it is not known whether CD8 a l p heterodimers similarly bind to class I molecules.
Until now there has been only limited information as to whether the two CD8 isoforms found on transfected cells were also expressed under physiological conditions. For example, murine epithelial lymphocytes, now known to express TcRy/6 to a large extent, were found to be Ly-2+/Ly-3- [43]. In addition, a murine CD4+CD8+T cell clone was reported to express Ly-2 in the absence of Ly-3 molecules [39]. In man,TcRy/6 thymocytes were reported to express CD8 a/a homodimers [41]. In addition, circulating CD8+ T lymphocytes expressingTcR y/6 were found to be unreactive with mAb CD8.2 [40]. Structural analysis by means of immunoprecipitation and SDS-PAGE demonstrates that CD8 a/a+ T lymphocytes express two CD8 a proteins of 32 kDa molecular weight as
Besides contributing to cell adhesion, CD8 is believed to function in signaling events [25-271. When Tlymphocytes expressing either CD8 a alone or CD8 a plus CD8 p were compared, it was found that the CD8 isoforms serve different functions.Thus, in contrast to CD8 a@-expressing cells, CD3-mediated cytotoxicity of a CD8 d a T cell clone was not inhibited by CD8 mAb and, moreover, CD3/CD8 cross-linking did not lead to proliferation of this clone.The susceptibility towards CD8 mAb-induced inhibition was explained by the fact that CD8 mAb perturbate either a negative signal or, alternatively, inhibit the positive signaling by CD8 mediated through CD3/CD8 interaction [25-281. One might, therefore, conclude that C D 8 d a
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homodimers are not involved in CD3-mediated signal- 18 Norment, A. M. and Littman, D. R., EMBO J. 1988. 7: 3433. ing. 19 Shiue, L., Gorman, S. D. andParnes, J. R., J. Exp. Med. 1988. Interestingly, tbose lymphocytes expressing CD8 a but not 168: 1993. CD8P either do not express CD3, or express CD3 in 20 Gorman,S. D., Sun,Y. H., Zamoyska, R. and Parnes, J. R., J. Irnrnunol. 1988. 140: 3646. association with a TcRylG, or coexpress C D 8 and CD4 when being TcRdP+. It seems, therefore, likely that 21 DiSanto, J. F!, Knowles, R. W. and Flomenberg, N., EMBO J. 1988. 7: 3465. C D 8 d a homodimers are unrelated to MHC class I restric'22 DiSanto, J. F!, SmaU,T. N., Dupon, B., Flomenberg, N. and tion in CD8+ 'RR dP+Tlymphocytes. Knowles, R. W., in McMichael, A. J. et al. (Eds.), Lecrcocyte Typing Ill, Oxford University Press, Oxford 1987, p. 210. As demonstrated earlier, supertransfection of CD8 a cDNA into TcRdB transfected cells resulted in the a u p 23 Norment, A. M., Salter, R. D., Rarham, F!, Engelhard,V. E. and Littman, D. R., Nature 1988. 336: 79. mentation of the response towards antigen [44,45].This effect was explained by the adhesion and signaling function 24 Rosenstein,Y., Ratnofsky, S., Burakoff, S. J. and Herrmann, S. H., J. Exp. Med. 1989. 169: 149. of CD8 a suggesting that C D 8 P is less important for CD8 25 Emmrich, F., Strittmatter, U. and Eichmann, K., Proc. Natl. signaling. The present studies performed on natural CD8 Acad. Sci. USA 1986. 83: 8298. molecules do not support such a view. Recent studies 26 Samstag,Y., Emmrich, F. and Staehelin,T., Proc. Natl. Acad. employing natural and truncated forms of CD4 and CD8 a Sci. USA 1988. 85: 9689. molecules, respectively, which suggest that CD8 a only 27 Van Seventer, G. A.,Van Lier, R. A. W., Spits, H., Ivanyi, F! and Melief, C. J. M., in Knapp, W. et al. (Ecis.), Lewocyre exhibits cell adhesion [46]would be in line with the present Typing Iv Oxford University Press, Oxford 1989, p. 206. data. 28 Quinones, R. R., Segal, D. M., Henkart, F!, Rerez, R.and Received April 2, 1991; in revised form April 26, 1991.
5 References 1 Reinherz, E. L. and Schlossman, S. F., Cell 1980. 19: 821. 2 Jitsukawa, S., Faure, F., Lipinski, M. ,Triebel, F. and Hercend, T., J. Exp. Med. 1987. 166: 1192. 3 Groh,V., Porcelli, S., Fabbi, M., Lanier, L. L., Picker, L. J., Anderson, T., Warnke, R. A., Bahn, A. K., Strominger, J. L. and Brenner, M. B., J. Exp. Med. 1989. 169: 1277. 4 Hercend, T., Reinherz, E. L., Meuer, S. C., Schlossman, S. F. and Ritz, J., Nature 1983. 301: 158. 5 Meuer, S. C., Schlossman,S. F. and Reinherz, E. L., Proc. Natl. Acad. Sci. USA 1982. 79: 4395. 6 Brenner, M. B., McLean, J., Dialynas, F!, Strominger, J. L., Smith, J. A., Owen, F. L., Seidman, J. G., Ip, S., Rosen, F. and Krangel, M. S., Nature 1986. 322: 145. 7 Moingeon, F!,Ythier, A., Goubin, G., Faure, E, Noville, A., Delmon, L., Rainaud, R., Forestier, E, Dafoss, E, Bohuon, C. and Hercend, T., Nature 1986. 323: 638. 8 Matis, L. A., Cron, R. and Bluestone, J. A. ,Nature 1987.330: 262. 9 Vandekerckhove, B. A. E., Datema, G.,Koning, F., Goulmy, E., Persijn, G. G. ,Van Rood, J., Claas, F. H. J. and DeVries, J. E., J. Immunol. 1990. 144: 1288. 10 Vidovic, D., Roglic, M., McKune, K., Guerder, S., MacKay, C. and DembiC, Z., Nature 1989. 340: 646. 11 BoMeville, M., Ito, K., Krecko, E. G., Itohara, S., Kappes, D., Ishida, I., Kanagawa, O., Janeway, C. A., Murphy, D. B. and Tonegawa, S., Proc. Natl. Acad. Sci. USA 1989. 86: 5928. 12 Porcelli, S., Brenner, M. B., Greenstein, J. L., Balk, S. F?, Rrhorst, C. and Bleicher, F! A., Nature 1989. 341: 447. 13 Herberman, R. B. and Ortaldo, J. R., Science 1981.214: 24. 14 Snow, F! M. and Terhorst, C., J. Biol. Chem. 1983. 258: 14675. 15 Littman, D. R.,Thomas,Y., Maddon, I? J., Chess, L. and Axel, R., Cell 1985. 40: 237. 16 Kavathas, I?, Sukhatme,V.F!, Herzenberg, L. A. and Parnes, J. R., Proc. Natl. Acad. Sci. USA 1984. 81: 7688. 17 Johnson, F! , lmmunogenetics 1987. 26: 174.
Gress, R. E., J. Immunol. 1989.142: 2200. 29 Veillette, A., Bookman, M. A., Horak, E. M. andBolen, J. B., Cell 1988. 55: 301. 30 Barber, E. K., Dasgupta, J. D., Schlossman,S. F.,Trevillyan, J. M. and Rudd, C. E., Proc. Natl. Acad. Sci. USA 1989. 86: 3277. 31 Moebius, U., Manns, M., Hess, G., Kober, G., Meyer zum Biischenfelde, K.-H. and Meuer, S. C., Eur. J. Zmmunol. 1990. 20: 889. 32 Romain, F! L., Acuto, 0. and Schlossman, S. E, Methodr Enzymol. 1984. 108: 624. 33 Laemmli, U. K., Nature 1970. 227: 680. 34 Chirgwin,J. M., Przybyla, A. E., MacDonald, R.J. and Rutter, W. J., Biochemistry 1979. 18: 5294. 35 Davis, L. G., Dibner, M. D. and Battey, J. F. (Eds.), Basic methods in molecular biologj Elsevier, New York 1986, p. 143. 36 Schraven,B., Samstag,Y.,Altevogt, F! and Meuer, S. C., Nature 1990.344: 71. 37 Snow, M. I?, Keizer, G., Collogan, J. E. and Terhorst, C., J. Immunol. 1984. 133: 2058. 38 Snow, I?,Spits, H., DeVries, J. E. and Terhorst, C., Hybridoma 1983. 2: 187. 39 Gallagher, P. E, Fazekas de St. Groth, B. and Miller, J. E A. I!, Eur. J. Immunol. 1986. 16: 1413. 40 Terry,L. A.,DiSanto,J.I?andFlomenberg,N.,TissueAntigens 1989. 33: 100 (Abstract). 41 McDonald,H. R., Schreyer, M., Howe, R. C. and Bron, C., Eur. J. Immunol. 1990. 20: 927. 42 Blue, M. L., Daley, J. E, Levine, H. and Schlossman, S. F., J. Immunol. 1985. 134: 2281. 43 Parrott, D. M. V , n i t , C., MacKenzie, S., McI. Mowat, A., Davies, M. D. J. and Micklem, H. S., Ann. N.YAcad. Sci. USA 1983. 409: 3W. 44 Dembib, Z., Haas,W., Zamoyska, R., Parnes, J., Steinmetz, M. and Von Boehmer, H., Nature 1987.326: 510. 45 Letourneur, F., Gabert, J., Cosson, F!, Blanc, D., Davoust, J. and Malissen, B., Proc. Natl. Acad. Sci. USA 1990. 87: 2339. 46 Von Hoegen, I?, Miceli, M. C. and Parnes, J. R., lmmunobiology 1990. 151: 15Oa (Abstr.).
Ulrich Moebius., Gabriela Kober., Anne Lise GriscelW, Thierry Hercende and Stefan C. Meuer. Abteitung Angewandte Immunologie., Deutsches Krebsforschungszentrum, Heidelberg and DB artement de Biologie Celldaire~,Institut Gustave Roussy, Viejuif
Functionally distinct CD8 isoforms
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Expression of different 0 8 isoforms on distinct human lymphocyte subpopulations Human CD8+ lymphocyte subpopulationswere analyzed for their expression of CD8 a and CD8 p subunits. Investigations with uncloned peripheral blood lymphocytes as well as cloned human natural killer and Tcell subpopulations demonstrate that CD3- natural killer cells, Tcell receptor y/S, and CD4+CD8+ Tcell clones express exclusively CD8 a gene products. Structural analysis of CD8 molecules demonstrates that CD8 a+@-T lymphocytes surface express 75-kDa CD8 d a homodimers whereas CD8 d/3lymphocytes express concomittantly two CD8 isoforms of different molecular masses (67 kDa and 75 kDa, respectively). Peptide mapping of these latter two isoforms suggests that CD8 is expressed as da homodimers and a/fi heterodimers on CD8 cells. Importantly,we found that the two CD8 isoforms behave functionally different. Thus, in contrast to CD8 dp+/CD8 a/a+ T lymphocytes, cytolytic activity of CD8 a/P-/CD8 a/a+ T cell clones was not inhibited by anti-CD8monoclonal antibodies and the latter were not induced to proliferate following CD3/CD8 cross-linking.
1 Introduction
gene products can be expressed as homodimers on mouse Lcells. In contrast, surface expression of CD8 fi proteins The leukocyte differentiation antigen CD8 is expressed on required cotransfection of the CD8 a gene and was linked to various subpopulations of PBL: (a) a subset of the expression of CD8a molecules [20,21]. Homodimeric CD3+/'EcRdfi Tlymphocytes [l], (b) a subset of vs. heterodimeric configuration of human CD8 chains CD3+/'EcRy/6Tlymphocytes [2,3] and (c) a minor popu- expressed on L cells were distinguishable by means of one lation of CD3- NK cells [4]. CD8+/TcRd p Tlymphocytes particular anti-CD8 mAb, namely 2ST85H7, which does are known to recognize Ag in the context of MHC class I not react with CD8a transfectants [18, 19, 221. molecules and to exhibit cytotoxidsuppressor function [1,5]. In contrast, TcR y/S T lymphocytes were reported to CD8 was demonstrated to bind MHC class1 molecules predominantly exhibit non-MHC-restrictedcytotoxicactiv- directly [23,24]. Moreover, mAb directed at CD8 were ity [6,7], although some y/S Tcells were reported to reported to modulateT lymphocyte activation and effector recognize antigen in association with MHC class I mole- function either by inhibition or, alternatively, stimulation cules [8,9] as well as MHC class I-like molecules (CD1, Qa, depending on the experimental conditions employed TL; [lo-121). Finally, CD3- NK cells exhibit MHC- [25-281. CD8 has been reported, therefore, to be involved unrestricted cytolytic activity towards a number of primary in lymphocyte activation not only by mediating cell-to-cell tumors and tumor cell lines [4,13]. contact but also by transmitting functional signals. The recent finding that CD8 is intracellularly linked to the CD8 exists as a dimeric membrane glycoprotein composed tyrosine kinase p5dCkhas provided a rational basis for such of two disulfide-linkedsubunits.The isolated murine CD8 a a view [29,30]. and CD8 p subunits (Ly-2, Ly-3) have a molecular mass of 32kDa and 37kDa, respectively, and are encoded by In the present report we describe the characterization of separate genes.The human CD8 molecule was described as CD8 molecules expressed on TcR df3+Tlymphocytes, a dimer of two 34-kDa subunits [14]. Initially,only one CD8 TcRy/G+ Tcells and CD3- NK cells. Whereas TcRa/p+ molecule was identified in SDS-PAGE and since only the T cells expressboth CD8 a and CD8 f3 chains,TcRy/S T cells CD8 a gene [15,16] was known, human CD8 was believed and NK cells express only CD8 a as homodimners. Functo exist as a homodimer. After the identification of the tional analysis indicates that the C D 8 d a and CD8dp human CD8p gene [17], however, it became evident that forms, respectively, behave differently. Furthermore the both genes, CD8a and CD8p are expressed in CD8+ present data provide evidence that CD8 a and CD8 @ chains Tlymphocytes [18, 191 suggesting a heterodimeric s t r u ~ are coexpressed in two different dimeric isoforms on ture of this molecule. So far, CD8a and CD8p gene TcR a/@+Tcells. products have not been unequivocally defined at the protein level. 2 Materials and m e t W Transfection studies employing mouse and human CD8 a and CD8 (3 cDNA, respectively, have shown that the CD8 a
2.1 Cells and cell cnltnre conditions
Tcell clones were established from peripheral blood of healthy donors and from lymphocytic liver infiltrates of [I 94251 patients with chronic active B hepatitis, respectively. PBL were prepared by density centrifugation employing Ficoll* This work was supported by DFG-grant: Me 69314-1. Hypaque (Pharmacia, Freiburg, FRG). Liver-infiltrating thrreqmStefan C. Meuer, Abteilung Angewandte Immu- lymphocyteswere recovered as described [31]. Subsequentn w e , Iust. fiir Radiologie und Patkophysiologie, Deutsches ly, lymphocytes were stimulated with PHA (0.3 pg/ml, Kmhfmchungszentrum, Im Neuenheimer Feld 280, D-6900 Wellcome, Burgwedel, FRG} under limiting dilution conditons and individual clones were expanded as described Heidelberg, FRG 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
+
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U. Moebius, G. Kober, A. L. Griscelli et al.
[5,31]. PHA-activated T lymphocytes were generated by incubating PBL for 7 days in medium containing 0.3 pg/ml PHA.
Eur. J. Immunol. 1991. 21: 1793-1800
precipitation was performed for 4 h. Immunoprecipitates were washed, subjected to SDS-PAGE according to Laemmli [33] and visualized by autoradiography employing Kodak (Rochester, NY) X-Omat AR films.
2.2 mAb
CD3 mAb were OKT3 (purchased from American Type Culture Collection, Rockville, MD), RW28C8 (IgG1, kindly provided by Dr. E. Reinherz, Dana-Farber Cancer Institute, Boston, MA), and BMA030 (provided by R. Kurrle, Behringwerke, Marburg, FRG); CD4 mAb (12T4Dll) was provided by Dr. E. Reinherz. CD8 mAb were CD8.1 (AICD8.1, IgG1, fromour laboratory), CD8.2 (2ST85H7, IgGza), (338.3 (7PT3F9, IgG2,), CD8.4 (21Thy2D3, IgGI), all from Dr. E. Reinherz, CD8.5 (AICD8.2, IgG1) and CD8.6 (AICD8.3, IgG2,), both from our 1aboratory.TcR-specificmAb were BMA031 (IgG2b, R. Kurrle) and TcR61 (IgG1, TCell Sciences, Cambridge, MA). 2.3 Immunofluorescence analysis For indirect immunofluorescence,106 cells were incubated for 30 min with saturating concentrations of mAb (1 :1000 dilution of ascites fluid) in medium containing 5% human serum [31]. Subsequently,cells were washed and incubated with FITC-labeled goat-anti-mouse Ig Ab (Coulter, Hialeah, FL). For two-color immunofluorescenceanalysis, cells were incubated simultaneously with two different mAb followed by an incubation with a 1:40 dilution of two appropriate isotype-specific goat anti-mouse Ig Ab (Southem Biotechnology, Birmingham, AL) which were FITCand PE-labeled, respectively. Red (fluorescence 1) and green (fluorescence2) fluorescence was determined in a Coulter Epics Profile Cytofluorograph.
2.6 Northern blot analysis
RNA was extracted from 2 x 107-5 x lo7 Tcells according to the method of Chirgwin et al. [34]. Approximately 15 pg RNA was denatured employing 50% formamide, 18% formaldehyde in 1x Mops for 15 min at 55°C and was size-separated on 1% agarose gels employing 10% formaldehyde and 1x Mops as running buffer [35]. Subsequently, RNA was transferred to Gene Screen membranes (NEN, Dreieich, FRG) according to the manufacturer's instructions. About 100 ng of the specific gene probe was labeled by nick translation employing 750 kBq 32P-a-dCiT(NEN) and a nick translation kit (Boehringer Mannheim, Mannheim, FRG). Probes were a 1.3-kb EcoRI CD8a cDNA insert [15] and a 1.0-kb EcoRI CD8p cDNA insert [18], kindly provided by D. Littman, Stanford, CA, and a plasmid with inserted chicken actin cDNA. Filters were prehybridized in 50% formamide and hybridized overnight with 2 X lo6 cprdml labeled probe and 5 pg/ml salmon sperm DNA in prehybridization solution at 42°C. Filters were washed with 2 x SSC at room temperature (RT), 0.1 x S S C + l % SDS (65°C) and 0.1xSSC (RT) and exposed to Kodak XAR films for 1to 3 days. For a second hybridization with a different probe filters were washed in 100 m~ Tris, 10 mM EDTA, 1% SDS, pH 8.0, at 100°Cfor 20 min, prehybridized and hybridized as mentioned above. 2.7 Peptide mapping
After separation of immunoprecipitates, unfixed SDS gels were dried and exposed at -70 "C. Appropriate bands were excised. Peptide mapping employing Staphylococcus V8 CD3 mAb-induced cell mediated cytotoxicity was analyzed protease (Sigma) at 0.1 mg/ml was performed as described in standard Cr-release assays employing lo3 51Cr-labeled previously [36]. Daudi cells as targets [ 5 ] . Prior to addition of effector cells, labeled target cells were preincubated with CD3 mAb (OKT3) for 20 min. Tlymphocytes were preincubated in 2.8 Cell proliferation assay parallel with mAb CD8.5 (1 : lo00 dilution of ascites fluid) Wells of a 96-well flat-bottom plate (Nunc, Roskilde, and control mAb (14G3, same isotype as CD8.5), respec- Denmark) that were precoated overnight with a rabbit tively, for 20 min and added to target cells at an E/Tratio of anti-mouse Ig Ab (5 pg/ml, Dianova, Hamburg, FRG) 2/1. Following a 3-h incubation, released radioactivity was were incubated for 2 h with mAb BMA030 (300 ng/ml), or determined and % specific lysis was calculated according to CD8.4, or 12T4Dll (both 1: 10oO dilution of ascites fluid), the formula: or a combination of two mAb. Subsequently,7 x 104Tcells cpm - spontaneous release were added per well. After a 48-h incubation, cells were % Specific lysis = x loo maximum release - spontaneous release pulsed with 37 kBq [3H]dThd (NEN, 75 GBq/mmol) and harvested after an additional 24 h. 2.5 Immunoprecipitation and SDS-PAGE
2.4 Cytotoxicity assays
Cell surface labeling was performed as described previously [32]. Briefly, 4 x lo7 cells were labeled with 37 MBq lZI (816 MBq/pg, Amersham, Braunschweig, FRG) employing lactoperoxidase (Sigma, Munich, FRG) and glucose oxidase (Sigma). Cells were washed and lysed with 1% Triton X-100 (Sigma). After centrifugation at 10000 x g SN were subjected to immunoprecipitation. mAb used for immunoprecipitation were covalently coupled to protein A-Sepharose (Pharmacia) with dimethylpimelimidate (Sigma). BSA was added to cell lysates at 1mg/ml and lysates were precleared for 2 h with protein A-Sepharose (three times) followed by one additional preclearing step with rabbit anti-mouse Ig protein A-Sepharose. Specific
3 Results 3.1 Expression of Merent CD8 isoforms as defined by mAb
Recent cluster analysis of CD8 mAb employing CD8 transfectants expressing human CD8 a and CD8 p gene products showed that all CD8 mAb investigated,with the exception of one mAb (2ST85H7), recognized C D S d p heterodimeric as well as the CD8 d a homodimeric forms [22]. Since mAb 2ST85H7 was only reactive with Lcells co-transfected with CD8 a and CD8 p it was concluded that this mAb is specific for an epitope of the CD8 p chain or, alternatively, recognizes a composite epitope encoded by
Eur. J. Immunol. 1991.21: 1793-1800
Functionally distinct CD8 isoforms
both CD8 a and CD8 P. mAb 2ST85H7 is designated in the following as CD8.2. Additional mAb employed in this study which recognize CD8 cc in the absence of CD8 P are termed (338.1, CD8.3,CD8.4, (38.5 and CD8.6, respectively (see Sect. 2.2).
Table 1. Phenotype of human cloned lymphocytes Clones
"'1
0.31
10,9]
54
CD3
r
12dl
+ + + + + + + + + + + + + + +
5A2
To investigate whether the CD8 P-restricted epitope recognized by mAb CD8.2 is coexpressed with CD8a on all CD8+human PBL, two-color immunofluorescenceanalysis was performed. As shown in Fig. 1B for one out of four representative donors, a considerable number of CD8 a+ PBL (CD8.4+)were unreactive with mAb CD8.2 (7.5 out of 18.4%). Cells only reactive with mAb CD8.2 were not observed. Moreover, CD8.2 mAb reacts exclusively with CD3+T cells (Fig. 1D) whereas a fraction of GD3- T lymphocytes (NK cells) is stained with mAb CD8.3 (specificfor the CD8 a chain; Fig. 1C). Similarly, mAb CD8.2 is clearly unreactive with TcRCJP- Tcells (Fig. 1F) as well as TcRy/G+ cells (Fig. 1H). In contrast, surface expression of the CD8 a gene product (detectable with mAb CD8.3) is found in TcRa/P+ (Fig.lE), a fraction of TcRy/G+ cells (Fig. lG), and a proportion of CD3- cells (Fig. lC),
01
mAb OKT3 BMA031 TcRG CD4 CD8.1
JT9 C3M 3025 BMlA6 JT3 F5511CI F551IICI
SD7 7405 Bd Vtc
WT3166
WM2 WM3
remectivelv. It seems. t,.erefore. that exmession of 6 8 d P b; human Tlymphocytes is intimatiy linked to the presence of a TcR dp-CD3 complex. To investigate this point further, a series of cloned human CD8+ lymphocyte populations were analyzed with regard to their reactivity with mAb CD8.1 (a chain-specific) and mAb CD8.2, respectively. As shown in Table 1, all Tcell clones and CD3- NK cell clones listed were reactive with mAb CD8.1 (and mAb CD8.3-6, not shown). All CD8+ T cell clones expressing a TcR d p were reactive with mAb CD8.2. In marked contrast to TcRdP+ Tcell clones, TcR y/G+ T cell clones and CD3- NK clones were unreactive with mAb CD8.2. In addition to CD3- and the TcRy/G+
CD3
7 4 0 5 , (A) I r4 0)
LI
E 3 C 1
16
32
40
64
B M l A 6 , (A)
d
TcR-a6
4 0)
V
log fluorescence 1
-
.-
Figure 1. Itno-color immunofluorescence analysis. PBL were stained with pairs of mAb as indicated. Numbers inside the gated
areas of each histogram indicate the percentage of positive cells. Numbers on the histogram axis represent channel numbers of fluorescence.
log fluorescence Figure 2. Indirect immunofluorescence. Clones C3F2, 7405, BMlA6 and WM2 were stained with (A) negative control, (B) CD8.1 and (C) CD8.2, respectively. Fluorescence was determined on an Epics Profile Cytofluorograph by analyzing loo00 cells per sample.
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clones, two TcRa/B Tcell clones were stained with mAb CD8.1 but not with mAb CD8.2 (WM2; WM3). Interestingly, these Tcell clones were found to stably coexpress CD4 and CD8 molecules as confirmed by means of two-color immunofluorescence (not shown). It should be noted that identical culture conditions were employed for all Tcell clones described here.
11684 58
The reactivity of four representative NK and T cell clones with mAb CD8.1 and CD8.2, respectively, is shown in Fig. 2. T cell clone C3F2 (BR a@) reacted with both types of CD8 mAb. In contrast, clones 7405 (TcRy/6), BMlA6 (NK), and WM2 ( R R d P , CD4+CD8+)were reactive with mAb CD8.1 but unreactive with mAb CD8.2. Expression of CD8 a by clones7405, BMlA6 and WM2 was lower than by CD8+I'IbRa@+ Tcell clones as judged from mean channel immunofluorescence (Fig. 2).
116
+
-.
48
36
84
--
58
-
48 +
36 26
-
Figure 4. CD8 immunoprecipitation. Immunoprecipitates from clones C3M (2),WM2 (3) and PBL (4) performed with negative control Ab (1) and CD8.1(2,3,4,5) were analyzed by SDS-PAGE on a 10% gel under nonreducing (A) and reducing (B) condi-
3.2 Expression of CDS a-and CD8 p-specific mRNA Representative Tcell and NK cell clones reactive with tions. either both types of CD8 mAb or CD8.1 only were subjected to Northern blot analysis to investigate the expression of CD8a and CD8P mRNA. As shown in o n n n o n Fig. 3, T cell clone C3F2 (lanes 1and 5 ) expressed CD8 a0 0 0 0 0 0 as well as CD8 P-specific mRNA. The TcR y/i3 Tcell clone 200 7405 (lane 2), the NK cell clone BMlA6 (lane 3) and T cell clone WM2 (lane 4) were found to express only CD8 a but not C D S P mRNA. A human B lymphoblastoid cell line 97 (M7) with no reactivity to any CD8 mAb did not express CD8 a and CD8 P genes (lane 6). 3.3 Immnnoprecipitation of individual CD8 isofonus
To characterize structurally the CD8 molecules of Tlymphocytes expressing either CD8a mRNA alone or along with CD8 p-specific mRNA, representative clones were surface labeled with lZsIand subjected to immunoprecipitation and SDS-PAGE. CD8.1 immunoprecipitates from Tcell clone C3F2 (CD8a@) and from PBL resulted in two 1
2
3
4
5
6
CD8-CI 18 s-
C D 8- A 18s
-
Actin 185
-
Figure 3. Northem blot andysis.Tital RNA of clones C3m (1, 5), 7405 (2), BMlA6 (3), WM2 (4), and the B-LCL M7 (6) was size-separated, transferred to a membrane and repeatedly hybridized with cDNA probes specific for CD8 a, CDS p and actin respectively.
68
-
43
-
29
-
Figure 5. CD8 immunoprecipitation. Immunoprecipitates from PHA-activated T lymphocytes employing various CD8 mAb were analyzed by SDS-PAGE on a 10% gel under nonreducing wnditions.
characteristic bands of 75 and 67 kDa when investigated under nonreducing conditions (Fig. 4A, lanes 2 and 4). In marked conrast, immunoprecipitates from clone WM2 (CD8da) resulted in only one band of 75 kDa (Fig. 4A, lane 3). Analysis of CD8 immunoprecipitates under reducing conditions showed a broad band of 31/32 kDa in the case of Tcell clone C3F2 and PBL (Fig. 4B, lanes 2 and 4). In contrast,WM2 gave a single and sharper band of 32 kDa (Fig. 4B, lane 3). To investigate which of these CD8 molecules is recognized by mAb CD8.2 and to compare several other CD8aspecific mAb we performed immunoprecipitatlon experiments*As shown in Fig- 5 9 mAb cD8-2 PreciPitated Only the 67-kDa molecule from lYsates of PHAactivated T lymphocytes. Besides mAb CD8.1, three additional CD8a mAb precipitated the 67 kDa molecule in
Functionally distinct CD8 isoforms
Eur. J. Immunol. 1991.21: 1793-1800
addition to the 75-kDa molecule (mAb CD8.4, CD8.5, CD8.6). In contrast mAb CD8.3 precipitated mainly the 75-kDa molecule (Fig. 5 ) . Note that mAb CD8.4, CD8.5 and CD8.6 precipitated different amounts of the two CD8 isoforms. 3.4 Peptide mapping of individual CD8 h f o m
The finding that GD8 d a Tlymphocytes express only the 75-kDa CD8 protein and that the 67-kDa molecule is related to an epitope recognized by the CD8P-restricted mAb CD8.2 suggests that the 75-kDa and 67-kDa proteins represent two CD8 isoforms of different subunit composition. To analyze further this question, we performed one-dimensional peptide maps. CD8 immunoprecipitates were separated by SDS-PAGE under nonreducing conditions, individual bands were excised and further subjected to V8 protease digestion and SDS-PAGE. As shown in Fig. 6, peptide mapping of 75-kDa proteins that were derived from CDSdP (C3F2) and C D 8 d a (WM2) T cell clones, respectively, resulted in identical fragments (lanes 2 and 3). Identity of peptide fragments was observed either when reducing (Fig. 6, lanes 2 and 3) or nonreducing conditions (Fig. 6, lanes 4 and 5) were employed for V8 protease digestion and SDS-PAGE.This suggests that the 75-kDa molecule expressed on CD8 dB T lymphocytes consists exclusively of CD8 a proteins and that this molecule can be considered as an a/a homodimer. In contrast, when the 75-kDa (Fig. 6, lane 2) and 67-kDa CD8 molecules (Fig. 6, lane 1) from a representative CD8 a/f3 T cell clone were compared, different peptide patterns were observed. Thus, digestion of the 67-kDa molecule resulted in one peptide (Fig. 6, position “c”) that was not present following digesiton of the 75-kDa molecule. In addition, fragments at position “d” were clearly distinct. Importantly, some fragments seem to be common 1
29
2
4
3
5
-a
14 -
-b
6-
-d
18
-C
3-
Figure 6. V8 protease peptide map of 75-kDa and 67-kDa CD8 molecules. CD8 immunoprecipitates from clones C3F2 (1, 2, 4) and WM2 (3,s) were subjected SDS-PAGE (10% gel) under nonreducing conditions. Subsequently, 75-kDa (2, 3, 4, 5) and 67-kDa (1)molecnles were excised, digested withV8 protease and analyzed on a 14% -20% gradient gel under reducing (1,2,3) and nonreducing (4,5) conditions.
200
-
97
-
68
-
43
-
1797
Figure 7. CD8 immunoprecipitation. Immunoprecipitates from Tcell clone 3025 performed with mAb CD8.2 and CD8.3, respectively,were analyzedby SDS-PAGE (10%) under nonreducing (A) and reducing (B) conditions.
for both proteins, particularly two fragments at position “a” and one at position‘%”. This suggests that the 67-kDa molecule in part consists of CD8a protein but contains additional protein(s). It should be noted that those fragments found to be common for both CD8 molecules were present with less relative intensity followingdigestion of the 67-kDa molecule.
To investigate further the subunit composition of the 67-kDa isoform we addressed the question of whether this dimer can be separated into two subunits. To this end, we employed mAb CD8.2 that precipitates only the 67-kDa molecule (Fig. 7A). For control purposes, the 75-kDa CD8 molecule was immunoprecipitated from a CD8+ NK clone employing mAb CD8.3. As shown in Fig. 7B, under reducing conditions the former precipitate yielded two separate bands of 32 kDa and 30 kDa, respectively. In contrast, reduction of the 75-kDa protein resulted only in the 32-kDa protein (Fig. 7B). Identical results were obtained when five additional CD8 dB T cell clones were analyzed (not shown). We then performed peptide map analysis of the 32 and 30-kDa polypeptides (compare Fig. 7B, CD8.2). As shown in Fig. 8, when the 32- and 30-kDa subunits were subjected separately to V8 protease treatment two widely differing peptide patterns were observed (Fig. 8, lanes 2 and 3). Thus, peptides at positions “a” and “b” were derived from the 32-kDa (Fig. 8, lane not from the 30-kDa molecule (Fig. 8, lane 3). resence of peptides at position “c” and “e” was less pronounced in the case of the 32-kDa than for the 30-kDa protein. This may result from cross-contamination due to incomplete separation of the subunits in SDS s at position “d” seem to represe in both, the 32and the 30-kDa molecule. However, the presence of two bands at this position was only observed for the 32-kDa
1798
U. Moebius, G. Kober, A. L.Griscelli et al. 1
2
Eur. J. Immunol. 1991.21: 1793-1800
Tmble 3. Prolilferative responses following CD3/CD8 stimula-
3
tion
clmr 17402
29 -
18
-
14 -
-a
Medium
-b
a33
-c
cm+cD8 m 3 cD8
W'
am
+
17411
4
a
144 116
us
76 54 142
6280
118 104 5492
5066
ND
ND
6s
a) mAb linked to tissue culture plate. b) rH]dThd uptake in cpm.
Figure 8. V8 protease peptide map of 32-kDa and 30-kDa CD8 molecules. CD8 immunoprecipitated from T cell clone 3025 employing mAb CD8.3 (1) and CD8.2 (2,3) were purified by SDS-PAGE on a 10% gel under reducing conditions. The 32-kDa (1,2) and 30-kDa (3) molecules were subsequently subjected toV8 protease treatment followed by SDS-PAGE on a 14%-20% gel under reducing conditions.
protein (Fig. 8, lanes 1 and 2). Importantly, peptide mapping of 32-kDa proteins, either derived from the 67-kDa dimer (lane 2) or the 75-kDa dimer (lane l), resulted in identical bands. Results analogous to those shown in Fig. 8 were also obtained when PHA-activated PBL were analyzed. These data support the view that the 67-kDa CD8 dimer consists of a 32-kDa CD8a subunit and a second 30-kDa subunit of different structure.
3.5 Functional analysis of CDS molecules expressed on CDSda and CDSdfl lymphocytes CD8 molecules were reported to be involved in T lymphocyte activation [25-28].Therefore,we were interested in the functional roles of the two different CD8 isoforms. To investigate this point, we studied the effect of CD8 mAb on the cytolytic activity of representative cloned lymphocytes (Table 2).To this end,various effector cell populations were pretreated with mAb prior to incubation in standard
Tmble 2. Functional effects of mAb on cytolytic activities of cloned effector cell populations
15D2
mAb 2lrfhp5D7
l2"4Dll
CD8.S
cKY8.4
cD4
50.8*1 22.3 28.9
9.2 ND 40.2
1.2 23.2 T.8
ND
12.2
12.0
Clone
Medium
Wr-release assays. As shown, the cytolytic activity of the CD4+/CD8a/a+ T cell clone WM2 towards its specific allogeneic target was not inhibited by mAb CD8.4. In addition, theTcR y/S T cell clone 7405 and the NK cell clone BMlA6 were not blocked by mAb CD8.5 andor CD8.4. In contrast, both CD8 mAb were strongly inhibitory for the Tcell clone C3F2. This inability of CD8 mAb to inhibit CD8 a/a lymphocytes could have been due to the fact that CD8 a/amolecules on these cells do not interact with MHC class I. Alternatively, functional effects independent of this CD8/MHC interaction could have been held responsible for this observation. To address the latter question,we employed Daudi cells, an FcR+, MHC class I- B lymphoma cell line which is not recognized by this set of Tcell clones. Addition of CD3 mAb (OKT3), however, brings together effector and target cells through binding to FcR and CD3, respectively, and triggers cytolytic effector function in a dose-dependent fashion. As shown in Fig. 9, CD3-induced cytotoxicity of Tcell clone C3F2 (CD8 a@+) was inhibited by addition of mAb CD8.5. In contrast, cytotoxicity of Tcell clone WM2 (CD8da) could not be blocked. This difference in CD8 mAb-mediated inhibition was observed at different levels of CD3-induced cytotoxicity. Note that inhibition of T cell clone C3F2 was specific for CD8 mAb since a control mAb of the same isotype did not result in reduction of cytolytic activity. Cross-linking of CD3 and CD8 (or CD3 and CD4) molecules was previously reported to induce proliferation of Okl-3
mAb
-
CD8
3x107 3x107 3x107 1x108 1x108 1x108
C3F2
CD8 Contr. ~
CD8 Contr
o
c3F2 wh42 BMlA6 1405
a) Percent specific lysis.
ND
WM2
10
10
30
u)
so
o
10
20
ao
.o
so
% SpeClfIC lpls
13.1
ND ND
Figure 9. Inhibition of cytotoxicity by CD8 mAb. C3F2 and WM2 cells, respectively,were incubated with 51Cr-labeledDaudi cells at an EfT ratio of 2/1. OK73 was added at 3 x and 10-8-fold dilution of ascites fluid. mAb CD8.5 was employed to study the function of CD8. Contr.: unrelated isotype-matched mAb.
Eur. J. Immunol. 1991. 21: 1793-1800
peripheral blood Tlymphocytes [25,26]. As shown in Table 3 when CD8 a / p Tcell clones were stimulated with submitogenic concentrations of surface-bound CD3 mAb, addition of CD8 a-specific mAb CD8.4 induced a proliferative response. In contrast, the CD4+/CD8a/a+/CD8a/pTcell clone WM2 was not induced to proliferate by CD3 plus CD8 mAb. This Tcell clone was, however, activated when CD4 molecules were cross-linked with CD3. These studies indicate that the two different CD8 isoforms expressed on C D 8 d a and C D 8 a / p Tlymphocytes serve different functions.
4 Discussion
Functionally distinct CD8 isoforms
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75-kDa disulfide-linkedhomodimers on the cell surface. In contrast, “conventional” CD8+ T lymphocytes were found to clearly express CD8 in at least two different dimeric isoforms, namely of 75 kDa and 67 kDa, respectively.The existence of two forms of the CD8 molecule (75 kDa and 67 kDa) was previously reported by others [14, 371. However, both molecules were believed to represent a/a homodimers with different intrachain disulfide bonds [38]. The present data demonstrate that the 75-kDa and 67-kDa molecules are composed of different subunits and that both isoforms can be co-expressed on unstimulated T cells and T cell clones.
Two separate genes were reported to encode for the CD8 a and the CD8 p subunits, respectively [15,17,20], and CD8 was known to be expressed as an a l p heterodimer on CD8+ Tlymphocytes [14, 37, 381. The present report demonstrates that CD8 is also expressed as an a/a homodimer on particular subsets of human lymphocytes. Thus, TcR y/S+ Tcells, CD3- NK cells, and RRa//P+, CD4+, CD8+ T cell clones were only reactive with CD8 a-specificmAb but did not bind mAb CD8.2 (2ST75H7) which reacts with CD8 p or a composite epitope of the a and P chains of CD8.Analysis of CD8 transcripts in a series of cloned human lymphocyte populations confirmed that they expressed CD8 a- but not CD8 P-specific mRNA consistent with the exclusive presence of CD8 a/a homodimers. In contrast, expression of CD8 p was only found on T lymphocytes expressing a TcR a@.
Following V8 protease treatment of the 67-kDa molecule those fragments common for the 75-kDa CD8 a/a molecule were relatively less intense as one would have expected assuming an a / p heterodimeric nature with 1: 1ratio of the two subunits. This might be explained with a different susceptibility to proteolysis of the CD8 a protein perhaps depending on the presence of the CD8 p molecule. Alternatively, it indicates that both proteins are not equally distributed and therefore not only present as heterodimers. It cannot be excluded that the 67-kDa immunoprecipitate consists of different CD8 dimeric isoforms which were coprecipitatedby mAb CD8.2.When under such conditions a/a homodimers were present, additional CD8 f3 protein would have been derived from fi/p homodimers. Since the exact specificity of mAb CD8.2 is not known, this, possibilitycannot be exclude at present. Note that this mAb is unreactive in Western blot analysis.
Cells expressing CD8 a/a homodimers represent minor subpopulations of human lymphocytes. Thus, a fraction of peripheral blood as well as tissue R R d p Tcells were reported to be CD8+ [2, 3, 39-41]. Similarly, a minor fraction of CD3- NK cells express CD8 [4]. Circulating T lymphocytes coexpressing CD4 and CD8 were only found at low frequencies in peripheral blood of normal donors [42]. In contrast, TcR a@ T lymphocytes expressing CD8 a and CD8p represent the majority of CD8+ peripheral lymphocytes [22,32]. These Tcells recognize antigen in combination with MHC class I molecules [5].
With regard to the specificityof various CD8 mAb our data indicate that those mAb precipitating both the 75-kDa and the 67-kDa molecules bind to epitopes of the a chain present in both isoforms. In contrast, mAb (338.2 and CD8.3 are specific for epitopes encoded by the a/p heterodimer and the a/a homodimer, respectively. Both CD8 isoforms could be noncovalentlyassociated on the cell surface and, therefore, coprecipitated. mAb CD8.2 and CD8.3, respectively, might disrupt the association of both isoforms because of their reactivity with epitopes that are crucial for CD8 ala-CD8 a l p interactions.
Transfection studies have previously demonstrated that the CD8 a protein can be expressed as an a/a homodimer on mouse L cells whereas the surface expression of the f3 chain required cotransfeciton of the a chain [20,21]. CD8 molecules consisting exclusively of fi chains have not been observed so far.
Previous findings that CD8+ Tlymphocytes were MHC class I restricted strongly suggested that CD8 binds directly to class I molecules. This was formally proven by binding studies employing reconstituted vesicles and transfected cells [23,24]. Since these transfection studies were performed employing CD8a genes it is not known whether CD8 a l p heterodimers similarly bind to class I molecules.
Until now there has been only limited information as to whether the two CD8 isoforms found on transfected cells were also expressed under physiological conditions. For example, murine epithelial lymphocytes, now known to express TcRy/6 to a large extent, were found to be Ly-2+/Ly-3- [43]. In addition, a murine CD4+CD8+T cell clone was reported to express Ly-2 in the absence of Ly-3 molecules [39]. In man,TcRy/6 thymocytes were reported to express CD8 a/a homodimers [41]. In addition, circulating CD8+ T lymphocytes expressingTcR y/6 were found to be unreactive with mAb CD8.2 [40]. Structural analysis by means of immunoprecipitation and SDS-PAGE demonstrates that CD8 a/a+ T lymphocytes express two CD8 a proteins of 32 kDa molecular weight as
Besides contributing to cell adhesion, CD8 is believed to function in signaling events [25-271. When Tlymphocytes expressing either CD8 a alone or CD8 a plus CD8 p were compared, it was found that the CD8 isoforms serve different functions.Thus, in contrast to CD8 a@-expressing cells, CD3-mediated cytotoxicity of a CD8 d a T cell clone was not inhibited by CD8 mAb and, moreover, CD3/CD8 cross-linking did not lead to proliferation of this clone.The susceptibility towards CD8 mAb-induced inhibition was explained by the fact that CD8 mAb perturbate either a negative signal or, alternatively, inhibit the positive signaling by CD8 mediated through CD3/CD8 interaction [25-281. One might, therefore, conclude that C D 8 d a
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homodimers are not involved in CD3-mediated signal- 18 Norment, A. M. and Littman, D. R., EMBO J. 1988. 7: 3433. ing. 19 Shiue, L., Gorman, S. D. andParnes, J. R., J. Exp. Med. 1988. Interestingly, tbose lymphocytes expressing CD8 a but not 168: 1993. CD8P either do not express CD3, or express CD3 in 20 Gorman,S. D., Sun,Y. H., Zamoyska, R. and Parnes, J. R., J. Irnrnunol. 1988. 140: 3646. association with a TcRylG, or coexpress C D 8 and CD4 when being TcRdP+. It seems, therefore, likely that 21 DiSanto, J. F!, Knowles, R. W. and Flomenberg, N., EMBO J. 1988. 7: 3465. C D 8 d a homodimers are unrelated to MHC class I restric'22 DiSanto, J. F!, SmaU,T. N., Dupon, B., Flomenberg, N. and tion in CD8+ 'RR dP+Tlymphocytes. Knowles, R. W., in McMichael, A. J. et al. (Eds.), Lecrcocyte Typing Ill, Oxford University Press, Oxford 1987, p. 210. As demonstrated earlier, supertransfection of CD8 a cDNA into TcRdB transfected cells resulted in the a u p 23 Norment, A. M., Salter, R. D., Rarham, F!, Engelhard,V. E. and Littman, D. R., Nature 1988. 336: 79. mentation of the response towards antigen [44,45].This effect was explained by the adhesion and signaling function 24 Rosenstein,Y., Ratnofsky, S., Burakoff, S. J. and Herrmann, S. H., J. Exp. Med. 1989. 169: 149. of CD8 a suggesting that C D 8 P is less important for CD8 25 Emmrich, F., Strittmatter, U. and Eichmann, K., Proc. Natl. signaling. The present studies performed on natural CD8 Acad. Sci. USA 1986. 83: 8298. molecules do not support such a view. Recent studies 26 Samstag,Y., Emmrich, F. and Staehelin,T., Proc. Natl. Acad. employing natural and truncated forms of CD4 and CD8 a Sci. USA 1988. 85: 9689. molecules, respectively, which suggest that CD8 a only 27 Van Seventer, G. A.,Van Lier, R. A. W., Spits, H., Ivanyi, F! and Melief, C. J. M., in Knapp, W. et al. (Ecis.), Lewocyre exhibits cell adhesion [46]would be in line with the present Typing Iv Oxford University Press, Oxford 1989, p. 206. data. 28 Quinones, R. R., Segal, D. M., Henkart, F!, Rerez, R.and Received April 2, 1991; in revised form April 26, 1991.
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Gress, R. E., J. Immunol. 1989.142: 2200. 29 Veillette, A., Bookman, M. A., Horak, E. M. andBolen, J. B., Cell 1988. 55: 301. 30 Barber, E. K., Dasgupta, J. D., Schlossman,S. F.,Trevillyan, J. M. and Rudd, C. E., Proc. Natl. Acad. Sci. USA 1989. 86: 3277. 31 Moebius, U., Manns, M., Hess, G., Kober, G., Meyer zum Biischenfelde, K.-H. and Meuer, S. C., Eur. J. Zmmunol. 1990. 20: 889. 32 Romain, F! L., Acuto, 0. and Schlossman, S. E, Methodr Enzymol. 1984. 108: 624. 33 Laemmli, U. K., Nature 1970. 227: 680. 34 Chirgwin,J. M., Przybyla, A. E., MacDonald, R.J. and Rutter, W. J., Biochemistry 1979. 18: 5294. 35 Davis, L. G., Dibner, M. D. and Battey, J. F. (Eds.), Basic methods in molecular biologj Elsevier, New York 1986, p. 143. 36 Schraven,B., Samstag,Y.,Altevogt, F! and Meuer, S. C., Nature 1990.344: 71. 37 Snow, M. I?, Keizer, G., Collogan, J. E. and Terhorst, C., J. Immunol. 1984. 133: 2058. 38 Snow, I?,Spits, H., DeVries, J. E. and Terhorst, C., Hybridoma 1983. 2: 187. 39 Gallagher, P. E, Fazekas de St. Groth, B. and Miller, J. E A. I!, Eur. J. Immunol. 1986. 16: 1413. 40 Terry,L. A.,DiSanto,J.I?andFlomenberg,N.,TissueAntigens 1989. 33: 100 (Abstract). 41 McDonald,H. R., Schreyer, M., Howe, R. C. and Bron, C., Eur. J. Immunol. 1990. 20: 927. 42 Blue, M. L., Daley, J. E, Levine, H. and Schlossman, S. F., J. Immunol. 1985. 134: 2281. 43 Parrott, D. M. V , n i t , C., MacKenzie, S., McI. Mowat, A., Davies, M. D. J. and Micklem, H. S., Ann. N.YAcad. Sci. USA 1983. 409: 3W. 44 Dembib, Z., Haas,W., Zamoyska, R., Parnes, J., Steinmetz, M. and Von Boehmer, H., Nature 1987.326: 510. 45 Letourneur, F., Gabert, J., Cosson, F!, Blanc, D., Davoust, J. and Malissen, B., Proc. Natl. Acad. Sci. USA 1990. 87: 2339. 46 Von Hoegen, I?, Miceli, M. C. and Parnes, J. R., lmmunobiology 1990. 151: 15Oa (Abstr.).
