Eur. J. Immunol. 1992. 22: 1413-1419
Andreas FischO and Konrad Reske Institut fur Immunologie der Universitat Mainz, Mainz
Cell surface expression of rat invariant y chain
1413
Cell surface display of rat invariant y chain: detection by monoclonal antibodies directed against a C-terminal y chain segment* A series of 14 monoclonal antibodies (mAb) directed against the C-terminal part of the rat invariant y chain (amino acid 142-216) was generated using distinct fusion proteins that contain this y segment for immunization and hybridoma screening. Additional fusion proteins were prepared carrying discrete regions of the ychain. Employing these reagents confirmed that the obtained mAb do indeed recognize the C-terminal portion of the invariant chain, as demonstrated by Western blot analysis. All mAb established recognize epitopes present on the native y chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenocytes combined with two-dimensional gel electrophoresis. However, while the majority of the y chainspecific mAb precipitated y chain-containing polypeptide chain complexes in which immature, sialic acid-deficient and mature, terminally sialylated forms of the y chain were predominantly represented, a fraction of the antibodies preferentially precipitated the immature y forms. Cell surface binding of these two groups of mAb correlated with the immunoprecipitation data in that the former group of antibodies did bind to intact Lewis rat spleen cells, while essentially no binding was observed with the antibodies of the latter group. Double-fluorescence staining with the class 11-specific fluorescein isothiocyanate-conjugated mAb OX3 and 0x6, respectively, as well as a representative y chain-specific mAb visualized with phycoerythrin-coupled secondary antibody shows coexpression of class I1 determinants and the invariant chain at the cell surface.
1 Introduction
extending into the cytoplasm and the carboxy terminus exposed at the exoplasmic site.
MHC class I1 molecules are polymorphic heterodimeric glycoproteins expressed at the cell surface of antigenpresenting cells. Class I1 molecules are integral membrane proteins that function as peptide-binding structures [l]. Recognition of peptide-carrying complexes by the T cell antigen receptor leads to functional activation of T lymphocytes. Class 11 molecules are comprised of two noncovalently associated subunits, a heavy chain a (33-35 kDa), and a light chain p (25-27 kDa). Within intracellular membrane systems, a nonpolymorphic glycoprotein (31 kDa), referred to as the invariant Ii or y chain, is attached noncovalently to the ap heterodimer, as demonstrated in the mouse [2], human [3] and rat system [4-61. In contrast to the polymorphic a and fi chains y represents a type-I1 transmembrane protein [7-91. It exhibits an inverted membrane orientation with its N-terminal part
Oligomeric apy complexes are formed shortly after biosynthesis of the individual proteins in the rough endoplasmic reticulum [lo]. In contrast to MHC class I molecules that follow the constitutive secretory pathway straight t o the cell surface y chain-containing MHC class I1 molecules are retarded within a sub-membrane region of the cell as suggested by biochemical and electron microscopy data [ll-131. A sorting signal within the N-terminal part of the y chain appears to be required by the a0 heterodimer to reach this region [14-161. Intersection of the flow of biosynthetic vesicles carrying class I1 complexes and the endocytic route was shown to occur in this cellular region [ll-131. Within this acid post-Golgi compartment the y chain is assumed to be removed from the class I1 complex by proteolytic cleavage [17-191.
[I 102241
Based on in vitro studies, the endosomal cathepsins were suggested to play an important role in this process [20]. Consistent with these data a@heterodimers exposed at the cell surface have been shown to be devoid of associated y chain [21-241.
* @
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 311, C2. This work was performed in partial fulfilment of the requirements of a doctoral thesis.
Correspondence: Konrad Reske, Institut fur Immunologie der Universitat Mainz, Obere Zahlbacherstr. 67, W-6500 Mainz,
FRG Abbreviations: 0-Gal: Galactosidase transferase aa: Amino acid
GST
Glutathione-S-
0 VCH Verlagsgescllschaft mbH, D-6940 Wcinheim, 1992
Despite detachment of the ychain from the oligomeric polypeptide chain complex within the intracellular compartment, previous work indicated the occurrence of y at the cell surface [25-281. To explore more thoroughly cell surface expression of the y chain and its potential significance in antigen presentation, we decided to prepare mAb directed against the extracytoplasmic carboxy-terminal part of the rat invariant chain. Using this approach, a panel of y-specific mAb was established and their reactivity pattern assessed.
+
0014-2980/92/0606-1413$3.50 .25/0
1414
A. Fisch and K. Reske
2 Materials and methods 2.1 Animals Lewis rats and BALB/c mice were obtained from our breeding facilities. Rats were used between 8 and 12 weeks of age, mice were used at the age of 6 to 8 weeks for primary immunization. Two female NZW rabbits purchased from Savo (Kisslegg, FRG) were given a primary immunization at the age of 8 weeks. 2.2 mAb Mouse mAb directed against RT1.B (I-A homologous) region-dependent MHC class I1 polypeptides derived from hybridomas OX6 and 0 x 3 , were kindly provided by Dr. A. F. Williams (University of Oxford, Oxford) [29]. mAb OX6 recognizes a common Ia determinant while mAb OX3 detects a polymorphic epitope present on class I1 molecules of Lewis, Wistar and A 0 rats [30]. Antibodies were employed as concentrated culture supernatants pretitrated before use.
Eur. J. Immunol. 1992. 22: 1413-1419
potassium acetate solution. The corresponding proteins were recovered by excising the bands followed by electroelution. Vector pGEXy-Pvu700 was used to transform E. coli JM 109. Fusion protein synthesis was induced by addition of isopropylthiogalactoside to a final concentration of 0.1 mM. Bacteria were lysed according to standard procedures and the soluble fusion protein possessing GSTactivity was purified by passage over a glutathione-Sepharose affinity column (Pharmacia, Freiburg, FRG). After extensive washing with PBS, 0.1% Triton X-100, the attached fusion protein was eluted with elution buffer (5 mM glutathione, 50 mM Tris-HC1, pH 8.0).
2.5 Preparation of xenoantiserum K8 directed against rat invariant y chain Two NZW rabbits were immunized by S.C. injection of 200 pg of pEXy-Pvu700 in PBYCFA. Two booster immunizations were performed administering 100 pg fusion protein in PBYIFA at intervals of 4 weeks. Blood was collected 10 days following each booster immunization.
2.3 Construction of recombinant plasmids Two procaryotic expression vectors were used for expression of fusion proteins containing distinct stretches of sequence of the rat invariant y chain (Fig. 1). Plasmids of the pEX series allow expression of cloned cDNA as fusion protein carrying the 117-kDa bacterial enzyme P-galactosidase @-Gal) [31]. In addition, the pGEX-3X vector was used to produce fusion proteins comprising an enzymatically active fragment of glutathione S transferase (GST; 26 kDa; [32]). cDNA coding for the entire sequence of the rat invariant y chain (clone 34/3) [33] was inserted into the Eco RI cloning site of pEX-2. The recombinant vector pEX-y served for further manipulation. Using this construct a 750-bp segment was deleted by cleavage with Sty I. The resulting plasmid pEXy-Sty200 contains sequence information coding for the 62 N-terminal amino acids (aa) of the rat invariant y chain. Two additional y fragments generated by digesting the y cDNA with Pvu I1 (Pvu400 and Pvu700) were both introduced into the Sma I site of pEX-3 yielding pEXyPvu400 and pEXy-Pvu700, respectively. Furthermore, the 3' fragment Pvu700 was also cloned into the Sma I polylinker region of the plasmid pGEX-3X resulting in pGEXyPvu700.The various recombinant vectors were employed to transform bacteria and the fusion proteins generated were isolated as described below and used for immunization and analytical studies. 2.4 Purification and isolation of fusion proteins The recombinant plasmids of the pEX series were introduced into E. coli pop2136 bacteria which allow plasmid amplification at low temperature and induction of fusion protein synthesis at 42 "C. Fusion proteins synthesized were recovered from bacterial lysates by preparative electrophoresis on a 5% polyacrylamide SDS gel. Protein bands were visualized by incubating the gel in ice-cold 1N
2.6 Generation of mAb directed against the C-terminal region of rat invariant y chain BALB/c mice were immunized three times with 20 pg of pGEXy-Pvu700. The fusion protein was applied i.p. in PBS/CFA and PBS/IFA, respectively, at intervals of 4 weeks. The final boost was given 2 months after the last immunization by i.p. and i.v. injection of 20 pg fusion protein in PBS. Three days later the spleen was removed and splenocytes were used for subsequent fusion. Sp2/0-Ag14 myeloma cells and immune splenocytes were fused in 49.3% (w/w) PEG 1000 and 3.65% (w/w) DMSO in IMDM at a ratio of 1: 1 [34, 351. The cell mixture was seeded into the wells of microtiter plates at a concentration of 5 x lo4spleen cells/well.The cultures were maintained in medium supplemented with 20 U/ml human recombinant IL-6 (Boehringer Mannheim, Mannheim, FRG). Culture supernatants were screened for the presence of y chainspecific antibodies by ELISA as described below. Specific antibody-producing hybridomas were cloned three times by limiting dilution. Positive clones were expanded and culture supernatant was collected and concentrated by ammonium sulfate precipitation. 2.7 Characterization of mAb
ELISA were performed in 96-well immunoplates (Nunc, Wiesbaden, FRG) using a total volume of 50 pUwe11 except for the blocking and washing where 200 pl/well were employed. Plates were coated with 1 pg/ml pEXy-Pvu700 and pEXy-Pvu400, respectively, to allow screening for y-reactive mAb.To prevent nonspecific binding, the culture wells were incubated with 2% BSA in PBS for 8 h at 4°C. After washing the wells twice with PBS, 0.1% Tween 20, hybridoma supernatants were added and incubated for 30 min at 37 "C. Following three washes peroxidase-conjugated anti-mouse Ig antibodies were added and incubated
Eur. J. Immunol. 1992. 22: 1413-1419
at 37°C for 30min. Following three additional washes, substrate solution containing 2 pg/ml 2.2' azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma, St. Louis, MO) and 0.3 pl/ml30% H202 in 10 mM sodium citrate buffer, pH 4.5, was added and allowed to react at room temperature for 15 min. Staining was recorded at 414 nm in an Immunoreader NJ-2000 (Nunc). To determine the subclass of the y chain-specific mAb, we established a protocol essentially involving the steps of the ELISA. Briefly, following incubation of the hybridoma supernatants within the fusion protein-coated wells subclass specific antisera conjugated with peroxidase (Camon, Wiesbaden, FRG) were added and binding was assessed by the staining procedure outlined above.
2.8 Biosynthetic radiolabeling, immunoprecipitation and 2-D gel electrophoresis Metabolic labeling of Lewis rat splenocytes was performed essentially as described in [26]. Radiolabeled Ig and nonspecifically adhering material were removed from NP40 cell lysates by incubation with normal rat serum (NRS, 100 pl/ml extract) for 1 h, followed by the addition of 200 p1 of 10% Pansorbin (Calbiochem, La Jolla, CA) for an additional 30 min. Specific immunoprecipitates were obtained by mixing the precleared detergent lysate with the respective mAb and pre-equilibrated protein A-Sepharose CL-4B (Biochrom, Berlin, FRG). Bound immunocomplexes were eluted by brief sonication in 2 x 30 pl IEF sample buffer and were analyzed by 2-D PAGE (first-dimension IEF; second dimension SDS-PAGE). Gels were soaked in Amplify (Amersham Int., Amersham, GB) dried under vacuum and exposed to Kodak X-OMAT AR film at - 70 "C for autoradiography.
2.9 Western blot analysis
Purified fusion proteins or crude bacterial lysates were separated on a 5% polyacrylamide gel under reducing conditions. Following incubation for 30 min in transfer buffer (48 mM Tris, 39 mM glycine, 20% (dv) methanol, pH 9.2) proteins were blotted onto a nitrocellulose membrane (Schleicher und Schull, Dassel, FRG) equilibrated in transfer buffer using a semi-dry electrophoretic transfer cell (Trans-Blot SD, Bio-Rad, Richmond, CA). Residual binding sites were blocked by incubating the membrane with 2% BSA in PBS overnight at 4°C. Following incubation with the respective culture supernatants diluted 1: 10 in PBS and following two washing steps in washing buffer (0,lYo Tween 20 in PBS) a peroxidase-conjugated antimouse Ig antibody (Sigma) was added (1 : 500 in PBS) and the membrane was incubated for another 2 h. After three final washes the membrane was placed in 200ml PBS. a-Chloronaphthol (150 mg) solubilized in 40 ml methanol and 130 ~1 30% H202 were added. Following a 15 min incubation at room temperature (RT) the membrane was washed with distilled water and dried at RT.
Cell surface expression of rat invariant y chain
1415
2.10 Flow cytometry Splenocytes of a Lewis rat depleted of erythrocytes (1 x 106/sample) were incubated for 30min at 4°C with the y chain-reactive mAb and antibody binding was detected using FITC-labeled goat anti-mouse IgG F(ab')2 (Medac, Hamburg, FRG). For double-fluorescence staining representative y chain-reactive mAb R G l l and RGlO (both of IgG2, subtype) as well as FITC-coupled mAb OX6 and 0 x 3 , respectively, (both of IgGl subtype) were employed. The medium used for all incubations was PBS containing 2% FCS and 2% NRS (both heat inactivated) including 0.1Y0 sodium azide. Two hundred microliters medium per sample was used. After washing two times in a volume of 1 ml, cells were incubated with a PE-conjugated antibody recognizing specifically mouse IgGz,. Following two subsequent washings and addition of 1 pg/ml of propidium iodide, cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
3 Results 3.1 Development of mAb against rat invariant y chain A glutathione S-transferase fusion protein (pGEXyPvu700) comprising the C-terminal part (aa 142-216) of the invariant y chain of the Lewis rat was prepared by subcloning (see Sect. 2.4) combined with affinity purification and was used to immunize BALBk mice. Splenocytes of these mice were subjected to subsequent somatic hybridization following standard procedures. Nearly 5000 proliferating hybridoma lines were generated. Culture supernatants were tested in an antibody-capture ELISA. P-Gal fusion protein pEXy-Pvu700 encompassing the same C-terminal segment of rat y chain (aa 142-216) and pEXy-Pvu400 containing another y segment (aa 15-140), used as a control for nonspecific binding, were prepared and employed to coat the wells of the ELISA plates. Fourteen independent positive hybridoma lines were identified and were cloned three times by limiting dilution. Isotype analysis revealed that roughly half of the clones, namely RG1-8, secrete antibodies of the IgGl subtype while the remaining clones RG9-14 produce IgG2, antibodies.
3.2 Assessment of the reactivity pattern of the mAb generated To verify that the established mAb are indeed directed against the C-terminal part of rat invariant y chain, several fusion proteins containing discrete segments of the y sequence (Fig. 1) were constructed and used in Western blot analysis. As shown for four representative clones in Fig. 2, all mAb recognize the fusion proteins carrying the Cterminal segment of they chain pEXy-Pvu700 (aa 142-216; lane 1) as well as the entire y chain pEXy (aa 1-216; lane 3). However they do not show any detectable reactivity with fusion proteins containing the middle stretch pEXy-Pvu400 (aa 15-140; lane 2) or the N-terminal piece pEXy-Sty200 (aa 1-62; lane 4) of the y chain sequence. Coomassie blue staining of a representative filter in the absence of mAb is shown for comparison.
1416
Eur. J. Immunol. 1992. 22: 1413-1419
A. Fisch and K. Reske mil
I
I
I
I
stvl
stvl
I
rat ?
Andreas FischO and Konrad Reske Institut fur Immunologie der Universitat Mainz, Mainz
Cell surface expression of rat invariant y chain
1413
Cell surface display of rat invariant y chain: detection by monoclonal antibodies directed against a C-terminal y chain segment* A series of 14 monoclonal antibodies (mAb) directed against the C-terminal part of the rat invariant y chain (amino acid 142-216) was generated using distinct fusion proteins that contain this y segment for immunization and hybridoma screening. Additional fusion proteins were prepared carrying discrete regions of the ychain. Employing these reagents confirmed that the obtained mAb do indeed recognize the C-terminal portion of the invariant chain, as demonstrated by Western blot analysis. All mAb established recognize epitopes present on the native y chain, as revealed by immunoprecipitation analysis using nonionic detergent extracts of metabolically labeled Lewis rat splenocytes combined with two-dimensional gel electrophoresis. However, while the majority of the y chainspecific mAb precipitated y chain-containing polypeptide chain complexes in which immature, sialic acid-deficient and mature, terminally sialylated forms of the y chain were predominantly represented, a fraction of the antibodies preferentially precipitated the immature y forms. Cell surface binding of these two groups of mAb correlated with the immunoprecipitation data in that the former group of antibodies did bind to intact Lewis rat spleen cells, while essentially no binding was observed with the antibodies of the latter group. Double-fluorescence staining with the class 11-specific fluorescein isothiocyanate-conjugated mAb OX3 and 0x6, respectively, as well as a representative y chain-specific mAb visualized with phycoerythrin-coupled secondary antibody shows coexpression of class I1 determinants and the invariant chain at the cell surface.
1 Introduction
extending into the cytoplasm and the carboxy terminus exposed at the exoplasmic site.
MHC class I1 molecules are polymorphic heterodimeric glycoproteins expressed at the cell surface of antigenpresenting cells. Class I1 molecules are integral membrane proteins that function as peptide-binding structures [l]. Recognition of peptide-carrying complexes by the T cell antigen receptor leads to functional activation of T lymphocytes. Class 11 molecules are comprised of two noncovalently associated subunits, a heavy chain a (33-35 kDa), and a light chain p (25-27 kDa). Within intracellular membrane systems, a nonpolymorphic glycoprotein (31 kDa), referred to as the invariant Ii or y chain, is attached noncovalently to the ap heterodimer, as demonstrated in the mouse [2], human [3] and rat system [4-61. In contrast to the polymorphic a and fi chains y represents a type-I1 transmembrane protein [7-91. It exhibits an inverted membrane orientation with its N-terminal part
Oligomeric apy complexes are formed shortly after biosynthesis of the individual proteins in the rough endoplasmic reticulum [lo]. In contrast to MHC class I molecules that follow the constitutive secretory pathway straight t o the cell surface y chain-containing MHC class I1 molecules are retarded within a sub-membrane region of the cell as suggested by biochemical and electron microscopy data [ll-131. A sorting signal within the N-terminal part of the y chain appears to be required by the a0 heterodimer to reach this region [14-161. Intersection of the flow of biosynthetic vesicles carrying class I1 complexes and the endocytic route was shown to occur in this cellular region [ll-131. Within this acid post-Golgi compartment the y chain is assumed to be removed from the class I1 complex by proteolytic cleavage [17-191.
[I 102241
Based on in vitro studies, the endosomal cathepsins were suggested to play an important role in this process [20]. Consistent with these data a@heterodimers exposed at the cell surface have been shown to be devoid of associated y chain [21-241.
* @
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 311, C2. This work was performed in partial fulfilment of the requirements of a doctoral thesis.
Correspondence: Konrad Reske, Institut fur Immunologie der Universitat Mainz, Obere Zahlbacherstr. 67, W-6500 Mainz,
FRG Abbreviations: 0-Gal: Galactosidase transferase aa: Amino acid
GST
Glutathione-S-
0 VCH Verlagsgescllschaft mbH, D-6940 Wcinheim, 1992
Despite detachment of the ychain from the oligomeric polypeptide chain complex within the intracellular compartment, previous work indicated the occurrence of y at the cell surface [25-281. To explore more thoroughly cell surface expression of the y chain and its potential significance in antigen presentation, we decided to prepare mAb directed against the extracytoplasmic carboxy-terminal part of the rat invariant chain. Using this approach, a panel of y-specific mAb was established and their reactivity pattern assessed.
+
0014-2980/92/0606-1413$3.50 .25/0
1414
A. Fisch and K. Reske
2 Materials and methods 2.1 Animals Lewis rats and BALB/c mice were obtained from our breeding facilities. Rats were used between 8 and 12 weeks of age, mice were used at the age of 6 to 8 weeks for primary immunization. Two female NZW rabbits purchased from Savo (Kisslegg, FRG) were given a primary immunization at the age of 8 weeks. 2.2 mAb Mouse mAb directed against RT1.B (I-A homologous) region-dependent MHC class I1 polypeptides derived from hybridomas OX6 and 0 x 3 , were kindly provided by Dr. A. F. Williams (University of Oxford, Oxford) [29]. mAb OX6 recognizes a common Ia determinant while mAb OX3 detects a polymorphic epitope present on class I1 molecules of Lewis, Wistar and A 0 rats [30]. Antibodies were employed as concentrated culture supernatants pretitrated before use.
Eur. J. Immunol. 1992. 22: 1413-1419
potassium acetate solution. The corresponding proteins were recovered by excising the bands followed by electroelution. Vector pGEXy-Pvu700 was used to transform E. coli JM 109. Fusion protein synthesis was induced by addition of isopropylthiogalactoside to a final concentration of 0.1 mM. Bacteria were lysed according to standard procedures and the soluble fusion protein possessing GSTactivity was purified by passage over a glutathione-Sepharose affinity column (Pharmacia, Freiburg, FRG). After extensive washing with PBS, 0.1% Triton X-100, the attached fusion protein was eluted with elution buffer (5 mM glutathione, 50 mM Tris-HC1, pH 8.0).
2.5 Preparation of xenoantiserum K8 directed against rat invariant y chain Two NZW rabbits were immunized by S.C. injection of 200 pg of pEXy-Pvu700 in PBYCFA. Two booster immunizations were performed administering 100 pg fusion protein in PBYIFA at intervals of 4 weeks. Blood was collected 10 days following each booster immunization.
2.3 Construction of recombinant plasmids Two procaryotic expression vectors were used for expression of fusion proteins containing distinct stretches of sequence of the rat invariant y chain (Fig. 1). Plasmids of the pEX series allow expression of cloned cDNA as fusion protein carrying the 117-kDa bacterial enzyme P-galactosidase @-Gal) [31]. In addition, the pGEX-3X vector was used to produce fusion proteins comprising an enzymatically active fragment of glutathione S transferase (GST; 26 kDa; [32]). cDNA coding for the entire sequence of the rat invariant y chain (clone 34/3) [33] was inserted into the Eco RI cloning site of pEX-2. The recombinant vector pEX-y served for further manipulation. Using this construct a 750-bp segment was deleted by cleavage with Sty I. The resulting plasmid pEXy-Sty200 contains sequence information coding for the 62 N-terminal amino acids (aa) of the rat invariant y chain. Two additional y fragments generated by digesting the y cDNA with Pvu I1 (Pvu400 and Pvu700) were both introduced into the Sma I site of pEX-3 yielding pEXyPvu400 and pEXy-Pvu700, respectively. Furthermore, the 3' fragment Pvu700 was also cloned into the Sma I polylinker region of the plasmid pGEX-3X resulting in pGEXyPvu700.The various recombinant vectors were employed to transform bacteria and the fusion proteins generated were isolated as described below and used for immunization and analytical studies. 2.4 Purification and isolation of fusion proteins The recombinant plasmids of the pEX series were introduced into E. coli pop2136 bacteria which allow plasmid amplification at low temperature and induction of fusion protein synthesis at 42 "C. Fusion proteins synthesized were recovered from bacterial lysates by preparative electrophoresis on a 5% polyacrylamide SDS gel. Protein bands were visualized by incubating the gel in ice-cold 1N
2.6 Generation of mAb directed against the C-terminal region of rat invariant y chain BALB/c mice were immunized three times with 20 pg of pGEXy-Pvu700. The fusion protein was applied i.p. in PBS/CFA and PBS/IFA, respectively, at intervals of 4 weeks. The final boost was given 2 months after the last immunization by i.p. and i.v. injection of 20 pg fusion protein in PBS. Three days later the spleen was removed and splenocytes were used for subsequent fusion. Sp2/0-Ag14 myeloma cells and immune splenocytes were fused in 49.3% (w/w) PEG 1000 and 3.65% (w/w) DMSO in IMDM at a ratio of 1: 1 [34, 351. The cell mixture was seeded into the wells of microtiter plates at a concentration of 5 x lo4spleen cells/well.The cultures were maintained in medium supplemented with 20 U/ml human recombinant IL-6 (Boehringer Mannheim, Mannheim, FRG). Culture supernatants were screened for the presence of y chainspecific antibodies by ELISA as described below. Specific antibody-producing hybridomas were cloned three times by limiting dilution. Positive clones were expanded and culture supernatant was collected and concentrated by ammonium sulfate precipitation. 2.7 Characterization of mAb
ELISA were performed in 96-well immunoplates (Nunc, Wiesbaden, FRG) using a total volume of 50 pUwe11 except for the blocking and washing where 200 pl/well were employed. Plates were coated with 1 pg/ml pEXy-Pvu700 and pEXy-Pvu400, respectively, to allow screening for y-reactive mAb.To prevent nonspecific binding, the culture wells were incubated with 2% BSA in PBS for 8 h at 4°C. After washing the wells twice with PBS, 0.1% Tween 20, hybridoma supernatants were added and incubated for 30 min at 37 "C. Following three washes peroxidase-conjugated anti-mouse Ig antibodies were added and incubated
Eur. J. Immunol. 1992. 22: 1413-1419
at 37°C for 30min. Following three additional washes, substrate solution containing 2 pg/ml 2.2' azino-bis-(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (Sigma, St. Louis, MO) and 0.3 pl/ml30% H202 in 10 mM sodium citrate buffer, pH 4.5, was added and allowed to react at room temperature for 15 min. Staining was recorded at 414 nm in an Immunoreader NJ-2000 (Nunc). To determine the subclass of the y chain-specific mAb, we established a protocol essentially involving the steps of the ELISA. Briefly, following incubation of the hybridoma supernatants within the fusion protein-coated wells subclass specific antisera conjugated with peroxidase (Camon, Wiesbaden, FRG) were added and binding was assessed by the staining procedure outlined above.
2.8 Biosynthetic radiolabeling, immunoprecipitation and 2-D gel electrophoresis Metabolic labeling of Lewis rat splenocytes was performed essentially as described in [26]. Radiolabeled Ig and nonspecifically adhering material were removed from NP40 cell lysates by incubation with normal rat serum (NRS, 100 pl/ml extract) for 1 h, followed by the addition of 200 p1 of 10% Pansorbin (Calbiochem, La Jolla, CA) for an additional 30 min. Specific immunoprecipitates were obtained by mixing the precleared detergent lysate with the respective mAb and pre-equilibrated protein A-Sepharose CL-4B (Biochrom, Berlin, FRG). Bound immunocomplexes were eluted by brief sonication in 2 x 30 pl IEF sample buffer and were analyzed by 2-D PAGE (first-dimension IEF; second dimension SDS-PAGE). Gels were soaked in Amplify (Amersham Int., Amersham, GB) dried under vacuum and exposed to Kodak X-OMAT AR film at - 70 "C for autoradiography.
2.9 Western blot analysis
Purified fusion proteins or crude bacterial lysates were separated on a 5% polyacrylamide gel under reducing conditions. Following incubation for 30 min in transfer buffer (48 mM Tris, 39 mM glycine, 20% (dv) methanol, pH 9.2) proteins were blotted onto a nitrocellulose membrane (Schleicher und Schull, Dassel, FRG) equilibrated in transfer buffer using a semi-dry electrophoretic transfer cell (Trans-Blot SD, Bio-Rad, Richmond, CA). Residual binding sites were blocked by incubating the membrane with 2% BSA in PBS overnight at 4°C. Following incubation with the respective culture supernatants diluted 1: 10 in PBS and following two washing steps in washing buffer (0,lYo Tween 20 in PBS) a peroxidase-conjugated antimouse Ig antibody (Sigma) was added (1 : 500 in PBS) and the membrane was incubated for another 2 h. After three final washes the membrane was placed in 200ml PBS. a-Chloronaphthol (150 mg) solubilized in 40 ml methanol and 130 ~1 30% H202 were added. Following a 15 min incubation at room temperature (RT) the membrane was washed with distilled water and dried at RT.
Cell surface expression of rat invariant y chain
1415
2.10 Flow cytometry Splenocytes of a Lewis rat depleted of erythrocytes (1 x 106/sample) were incubated for 30min at 4°C with the y chain-reactive mAb and antibody binding was detected using FITC-labeled goat anti-mouse IgG F(ab')2 (Medac, Hamburg, FRG). For double-fluorescence staining representative y chain-reactive mAb R G l l and RGlO (both of IgG2, subtype) as well as FITC-coupled mAb OX6 and 0 x 3 , respectively, (both of IgGl subtype) were employed. The medium used for all incubations was PBS containing 2% FCS and 2% NRS (both heat inactivated) including 0.1Y0 sodium azide. Two hundred microliters medium per sample was used. After washing two times in a volume of 1 ml, cells were incubated with a PE-conjugated antibody recognizing specifically mouse IgGz,. Following two subsequent washings and addition of 1 pg/ml of propidium iodide, cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
3 Results 3.1 Development of mAb against rat invariant y chain A glutathione S-transferase fusion protein (pGEXyPvu700) comprising the C-terminal part (aa 142-216) of the invariant y chain of the Lewis rat was prepared by subcloning (see Sect. 2.4) combined with affinity purification and was used to immunize BALBk mice. Splenocytes of these mice were subjected to subsequent somatic hybridization following standard procedures. Nearly 5000 proliferating hybridoma lines were generated. Culture supernatants were tested in an antibody-capture ELISA. P-Gal fusion protein pEXy-Pvu700 encompassing the same C-terminal segment of rat y chain (aa 142-216) and pEXy-Pvu400 containing another y segment (aa 15-140), used as a control for nonspecific binding, were prepared and employed to coat the wells of the ELISA plates. Fourteen independent positive hybridoma lines were identified and were cloned three times by limiting dilution. Isotype analysis revealed that roughly half of the clones, namely RG1-8, secrete antibodies of the IgGl subtype while the remaining clones RG9-14 produce IgG2, antibodies.
3.2 Assessment of the reactivity pattern of the mAb generated To verify that the established mAb are indeed directed against the C-terminal part of rat invariant y chain, several fusion proteins containing discrete segments of the y sequence (Fig. 1) were constructed and used in Western blot analysis. As shown for four representative clones in Fig. 2, all mAb recognize the fusion proteins carrying the Cterminal segment of they chain pEXy-Pvu700 (aa 142-216; lane 1) as well as the entire y chain pEXy (aa 1-216; lane 3). However they do not show any detectable reactivity with fusion proteins containing the middle stretch pEXy-Pvu400 (aa 15-140; lane 2) or the N-terminal piece pEXy-Sty200 (aa 1-62; lane 4) of the y chain sequence. Coomassie blue staining of a representative filter in the absence of mAb is shown for comparison.
1416
Eur. J. Immunol. 1992. 22: 1413-1419
A. Fisch and K. Reske mil
I
I
I
I
stvl
stvl
I
rat ?
