0 1992 Hanvood Academic Publishers Gmbh Printed in the United Kingdom
Autoimmunity, 1992, Vol. 11, pp. 171-177 Reprints available directly from the publisher Photocopying permitted by license only
DETECTION OF AN IDIOTOPE ON A HUMAN MONOCLONAL AUTOANTIBODY BY MONOCLONAL ANTI-IDIOTYPIC ANTIBODY AND ITS RELATIONSHIP TO EPSTEIN-BARR VIRUS-INDUCED AUTOIMMUNITY CARL0 GARZELLI, MARINA INCAPRERA, AGOSTINO BAZZICHI and GIUSEPPE FALCONE Department of Biomedicine, Section of Microbiology, University of Pisa, Italy
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
(Received June 7,1991; in finalform September 27,1991) We have recently described a human IgM monoclonal antibody (mAb), reactive with both self antigens, i.e., cytoskeleton filaments and smooth muscle, and Epstein-Barr virus (EBV)-induced nuclear antigen (EBNA), produced by EBV-transformed B lymphocytes isolated from a patient with infectious mononucleosis (IM). In order to achieve higher antibody secretion in culture supernatant, the mAb-producer cells were fused with ouabain-resistant mouse myeloma cells and a stable human-mouse heterohybrid, coded HY 5488, producing up to 80 pg/ml IgM mAb, was isolated after 4 cloning procedures. Purified HY 5844 mAb was used to immunize mice for the production of a murine anti-idiotypic mAb, which was used to probe the expression of the idiotope of HY 5488 mAb (Id 5488) in sera of IM patients and normal controls by ELISA. It was found that Id 5488 is expressed both in IM patients and normal controls, and that Id 5488 expression is significantly higher in IM patients' sera; furthermore, in IM sera a statistically significant correlation between Id 5488 expression and anti-cytoskeleton and anti-smooth muscle autoantibodies was found. It is suggested that at least part of EBV-induced IgM autoantibodies appearing during IM are secreted by B lymphocytes programmed to the production of "natural antibodies" bearing Id 5488-like idiotopes. KEY WORDS: Epstein-Barr virus, monoclonal autoantibody, idiotype.
INTRODUCTION Epstein-Barr virus (EBV), the agent of infectious mononucleosis (IM), is proposed to be involved in the etio-pathogenesis of some diseases with autoimmune components, such as rheumatoid arthritis, systemic lupus erythematosus and Sjogren syndrome'. IM itself is characterized by a transient occurrence of autoantibodies (autoAbs) of the IgM class to several antigens, including, to mention only the most frequently found, those against smooth muscle and cytoskeleton antigens of epithelial cells, appearing in over 70-80% IM, therefore, represents an appropriate model for the study of the mechanism(s) by which EBV induces autoimmunity and, possibly, for investigations on the relationships of EBV to autoimmune diseases. The mechanism(s) responsible for the appearance of EBV-induced autoAbs are not exactly known, even though virus-induced polyclonal B cell activation seems to play the major role; in fact, it is generally accepted that, upon infection, EBV-transformed B lymphocytes are triggered to express their antibody
Correspondence to: Dr Carlo Garzelli, Dipartimento di Biomedicina, Sezione di Microbiologia, Via San Zeno, 39, 56100 Pisa. Italy.
repertoire, including autoAbs'-''. Mechanisms other than virus-induced polyclonal B cell activation, however, have been also proposed. They include: (a) autoimmunization with cell antigens released by EBVinduced tissue damage3*"; and (b) immunization with EBV-encoded nuclear antigen (EBNA- 1) bearing molecular homology with normal tissue proteins". One approach to the study of the mechanism(s) of EBV-induced autoimmunity has been the investigation of human monoclonal autoAbs (mAb) secreted by EBV-transformed B lymphocytes: it has been shown that a large part of EBV-transformed B lymphocytes from normal individuals or patients with autoimmune diseases produce monoclonal IgM autoAbs that, when studied in detail, turned out to be polyspecific or reactive with antigens in multiple o r g a n ~ ~ . ' ' , ' ~in; this context, we have recently described a human monoclonal autoAb, designated CAT- 182-6-3, produced by EBV-transformed B lymphocytes isolated from a patient in the acute phase of IM, reactive with both cytoskeleton filaments and smooth muscle, as well as with EBNA14, raising the question of the relevance of this autoAb in EBVinduced autoimmunity. Since the role of autoAbs in autoimmunity can be usefully approached through idiotype studies, in that it is known that in mice and humans certain idiotypes are closely associated with particular autoimmune dis171
172
C. GARZELLI ET AL.
MATERIALS AND METHODS
specificity, assumed as Id 5488-negative (or minimally expressed), in 100 p l of carbonate-bicarbonate buffer, pH 9.6. The bound mouse antibody was detected using peroxidase-labeled goat anti-mouse IgG (7-chain specific; Sigma, USA). The positive wells showing reactivity with HY 5488 mAb, but not with polyclonal human IgM were cloned using the limiting dilution method. One hybridoma producing monoclonal IgGl antibody, coded HY 3D5-05, was obtained and finally produced into ascites in Balbk mice.
Production and purification of human monoclonal antibody
ELISA assay f o r HY 5488 idiotope (Id 5488) in human sera
The procedure for the isolation of EBV-positive CAT1E2-6-3 cells has been reported in detail elsewhere14. In this study, in order to achieve higher antibody production in culture supernatant, 2x10' CAT-1E2-6-3 cells were fused with 2x10' ouabain-resistant P3.X63.AG8.653 mouse myeloma cells and human -mouse heterohybrids were selected in ouabain -containing HAT medium, as previously described''. A stable human-mouse heterohybrid, coded HY 5488, was isolated after 4 limiting dilution cloning procedures. This hybrid produced up to 80 pglml of monoclonal IgM in spent culture medium with the same antibody reactivity as parent CAT-1 E2-6-3 EBV-positive lymphoblastoid cells. HY 5488 mAb was then purified from culture supernatant using an immunoaffinity column, which was prepared by coupling the IgG fraction of goat anti-human IgM ( p chainspecific; Sigma Co., USA) to CNBr-activated Sepharose 4B (Pharmacia Fine Chemical, Sweden). The adsorbed antibody was eluted with 0.1 M glycineHC1, pH 2.5. The purity of HY 5488 mAb was tested by SDS-polyacrylamide gel electrophoresis (PAGE), and two only bands having the apparent molecular weights of p- and k-chains were detected by silver stain.
Immunolon I1 ELISA plates were coated with l o o p 1 of 1 5 0 0 dilution in carbonate-bicarbonate buffer, pH 9.6, of HY 3D5-05 ascites at 4°C overnight. After 3 washes in phosphate buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tw), l o o p 1 of human sera diluted 1:lOO in PBS-Tw were added to the wells in duplicate and incubated at room temperature for 1 hr. After 3 washes in PBS-Tw, the wells were incubated for 1 hr at r.t. with l o o p 1 of 1:lOOO dilution in PBSTw of peroxidase-labeled goat anti-human IgM ( p chain specific; Sigma). After 5 final washes, plates were developed with 0.04% H202and 0.04% o-phenylenediamine in phosphate-citrate buffer, pH 5.0. The optical density at 492 nm was read using a Multiscan Microplate Reader (Flow Labs). Wells containing serial 2-fold dilutions of a.p. HY 5488 mAb, ranging from 5 ng/ml to 10 pg/ml, served as standards.
Preparation of anti-idiotypic monoclonal antibody
ELISA assay f o r Epstein-Barr virus viral capsid antigen (VCA) and nuclear antigen ( E B N A )
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
~ r d e r s l ~and , ' ~ only a limited number of Ig families are involved in autoimmunity", we have prepared a murine mAb directed against an idiotypic determinant o f the human CAT-1E2-6-3 mAb; furthermore, using this anti-idiotypic mAb we have studied the expression of the corresponding idiotope on IgM in the sera of normal individuals and patients with IM and its relationship to EBV-induced autoantibodies.
Balb/c female mice were immunized by 3 weekly, intraperitoneal injections of 50, 25, and 25 pg, respectively, of affinity purified (a.p.) HY 5488 mAb and by a last intravenous injection of 2 5 p g of a.p. mAb. Three days later, splenic lymphocytes were fused with P3.X63.AG8.653 mouse myeloma cells using 50% polyethylene glycol at neutral pH. The fused cells were plated in 96-well tissue culture plates and incubated at 37°C. Supernatants from outgrowing hybrids were screened for anti-HY 5488 and antihuman IgM antibody by ELISA, in which Immunolon I1 plates (Dynatech Laboratories, USA) were coated with 1 p g of either a.p. HY 5488 mAb or purified polyclonal IgM (Cappel Lab., USA) with irrelevant
IM sera Sera were obtained from patients with the clinical and haematological characteristics of JM, in whom the diagnosis had been confirmed by a positive Monospot test and by the presence of IgM antibodies to EBV viral capsid antigen (VCA).
Microtiter 96-well plates, coated with VCA or EBNA antigen, purified via monoclonal antibodies, were purchased from Dupont (Billerica, MA, USA). For assays, wells were incubated at r.t. for 1 hr with 20Opl of sera diluted 1:lOO in PBS-Tw. After 3 washes in PBS-Tw, the wells were incubated for 45 min at r.t. with 2 0 0 p l of 1:lOOO dilution in PBSTw of biotin-labeled goat anti-human IgM ( p chain specific, Sigma). After 3 additional washes as before, the wells were incubated for a further 45 min at r.t. with 200 pl of a 1 :1000 dilution in PBS-Tw of peroxidase-labeled anti-biotin (Sigma). After 5 final washes, plates were developed as described above.
IDIOTYPE EXPRESSION ON EBV-INDUCED AUTOANTIBODY
standard immunofluorescence techniques (not shown).
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
ELISA assay for anti-smooth muscle and anti-cytoskeleton autoantibody
On the ground of previous reports indicating that in IM anti-smooth muscle autoantibodies are mainly directed against tubulin subunits6, the method 0-f Shelanski, Gaskin and Cantor'' for the purification of this molecule was followed, with some modifications. Briefly, a sample of tissue from human stomach, obtained from a surgical specimen, was washed in cold PBS, minced using scissors, suspended (approximately 1 g/ml) in 0.1 M Hepes buffer, pH 6.8, containing 1 mM EGTA, 1 mM GTP and protease inhibitors, and then homogenized by using a Polytron blender at 4°C. After centrifugation at 4°C for 1.5 hr at 40,00Og, the pellet was discarded and the supernatant was mixed with an equal volume of 8 M glycerol in buffer and the resultant mixture was stirred at 37°C for 30 min. After centrifugation at 100,000g for 1.5 hr at 22"C, the precipitate, containing partially purified tubulin, was resuspended in carbonate-bicarbonate buffer, pH 9.6. ELISA plates were coated at 4°C overnight with 1 pg in 100pl of the tubulin preparation. For anti-cytoskeleton autoAbs assay, confluent microcultures of Hep-2 cells were grown in 96-well plates, washed three times with PBS, and then fixed with 0.1% glutaraldehyde in PBS for 5 min at r.t. The cells were washed once, incubated for 5 min at r.t. in 1% bovine serum albumine (BSA) in PBS, and then washed twice more before storage in PBS at 4°C. For both assays. wells were washed three times with PBS-Tw, incubated at r.t. for 1 hr with 100 pl of sera diluted 1:lOO in PBS-Tw, and then developed as described above. In preliminary experiments, the results of the above reported ELISA assays were shown to correlate with d
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and reactivity of HY 5488
human mAb Approximately one year after EBV transformation, CAT- I E2-6-3 cells, producing few micrograms of human monoclonal IgM per ml of spent culture medium, were fused with ouabain-resistant mouse myeloma cells and the resulting human-mouse hybrids were selected in HAT medium supplemented with ouabain. After 4 cloning procedures by limiting dilution, a stable heterohybrid, chosen on the basis of high IgM production, was isolated and expanded in culture medium. This hybridoma, coded HY 5488, secreted up to 80 pg/ml of monoclonal human IgM in spent culture medium. HY 5488 mAb was then produced by expanding the heterohybrid in 75 cm2 tissue culture flasks; the culture supernatant was concentrated approximately 10-fold by ultrafiltration and finally purified to homogeneity by affinity chromatography. The affinity-purified HY 5488 mAb showed the same antibody reactivity as parent CAT-lE2-6-3derived mAbI4, in that it reacted with both cytoskeleton filaments of cultured human epithelial cells and smooth muscle of frozen sections of human stomach by immunofluorescence, as well as with EBVencoded nuclear antigen (EBNA) by ELISA. Preparation of monoclonal anti-idotypic antibody Anti-idiotypic antibody was prepared by fusing spleen cells from Balb/c mice, immunized with a.p. HY 5488 mAb, with P3.X63.AG8.653 mouse myeloma cells.
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Figure 1 Specificity of HY 3D5-05 mAb for Id 5488. (Left), ELISA plate wells, coated with HY 3D5-05 mAb, received either serially diluted a.p. HY 5488 human mAb (circle) or the same concentrations of purified polyclonal IgM with irrelevant specificity, assumed as Id 5488-negative (triangle). (Right), E L S A plate wells, coated with either HY 3D5-05 ascites (circle) or P3.Xf~3.AG8.653 ascites (triangle), received serially diluted a.p. HY 5488
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
174
C. GARZELLI ET AL.
One successful fusion (480 wells) yielded a total of six outgrowing hybridomas that reacted with HY 5488 mAb by ELISA. After further testing and cloning, one of these, designated HY 3D5-05, was found to react with a.p. HY 5488 mAb, but not with purified polyclonal human IgM, assumed as Id 5488-negative (or minimally expressed) on the basis of their irrelevant specificity. The specificity of binding of HY 3D5-05 mAb to Id 5488 is further shown in Figure 1 (left), where it can be seen that wells coated with HY 3D505 murine mAb bind a.p. HY 5488 human mAb, but not the Id 5488-negative, purified polyclonal human IgM. To test the formal possibility that HY 5488 mAb had an affinity for mouse ascites antigens, the binding of a.p. HY 5488 mAb to wells coated with either HY 3D5-05 ascites or aspecific P3.X63.AG8.653 ascites was compared; as shown in Figure 1 (right), HY 5488 mAb strongly bound to wells coated with HY 3D5-05 mAb, as expected, and in a much lower extent to the wells coated with the aspecific ascites. By western-blotting, the HY 3D5-05 murine mAb failed to react with either heavy or light chains of the a.p. HY 5488 mAb blotted from SDS-PAGE gel run in reducing conditions (not shown).
relates with the titers of EBV-induced IgM autoantibodies. This analysis was performed by plotting the serum concentration of Id 5488-bearing IgM against the optical reading values of anti-VCA, anti-EBNA, anti-cytoskeleton, and anti-smooth muscle IgM antibody, respectively. Anti-VCA and anti-EBNA IgM antibody assays employed commercially available purified antigens; anti-cytoskeleton IgM autoantibodies were assayed by an ELISA assay employing glutaraldehyde-fixed Hep-2 cells; anti-smooth muscle IgM were assayed by an ELISA assay employing a semi-purified preparation of tubulin, i.e., the autoantigen against which the IM anti-smooth muscle autoantibodies are directed6. The results of this analysis are reported in Figure 3; as can be seen, no statistically significant correlation was found between Id 5488 expression and anti-VCA or anti-EBNA IgM antibodies (upper, left and right), whereas a statistically significant correlation was found between the Id 5488 expression and anti-cytoskeleton (P=0.005) and anti-smooth muscle IgM autoantibodies (P=0.003) (lower, left and right, respectively).
Detection of Id 5488-bearing IgM in normal and infectious mononucleosis sera
By fusing EBV-transformed human B lymphocytes with mouse myeloma cells, we have prepared stable
Twenty-nine sera from normal individuals, with no significant anti-VCA IgM activity, and 31 sera from patients in the acute phase of IM, as assessed according to the criteria reported in the Materials and Methods section, were tested by ELISA for the expression of Id 5488-bearing IgM. The results are reported in Figure 2. As can be seen, Id 5488 was detectable in all sera tested, but considerable quantitative differences were found among individuals in both tested groups; in particular, in normal individuals, the concentration of Id 5488-bearing IgM ranged between 1 and 20 pglml, whereas in IM patients the Id 5488-bearing IgM ranged between 4 and 40 pglml. The differences between the means (6.9 pg/ml and 15.2 pglml, for normal and IM patients, respectively) were statistically highly significant (P
Autoimmunity, 1992, Vol. 11, pp. 171-177 Reprints available directly from the publisher Photocopying permitted by license only
DETECTION OF AN IDIOTOPE ON A HUMAN MONOCLONAL AUTOANTIBODY BY MONOCLONAL ANTI-IDIOTYPIC ANTIBODY AND ITS RELATIONSHIP TO EPSTEIN-BARR VIRUS-INDUCED AUTOIMMUNITY CARL0 GARZELLI, MARINA INCAPRERA, AGOSTINO BAZZICHI and GIUSEPPE FALCONE Department of Biomedicine, Section of Microbiology, University of Pisa, Italy
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
(Received June 7,1991; in finalform September 27,1991) We have recently described a human IgM monoclonal antibody (mAb), reactive with both self antigens, i.e., cytoskeleton filaments and smooth muscle, and Epstein-Barr virus (EBV)-induced nuclear antigen (EBNA), produced by EBV-transformed B lymphocytes isolated from a patient with infectious mononucleosis (IM). In order to achieve higher antibody secretion in culture supernatant, the mAb-producer cells were fused with ouabain-resistant mouse myeloma cells and a stable human-mouse heterohybrid, coded HY 5488, producing up to 80 pg/ml IgM mAb, was isolated after 4 cloning procedures. Purified HY 5844 mAb was used to immunize mice for the production of a murine anti-idiotypic mAb, which was used to probe the expression of the idiotope of HY 5488 mAb (Id 5488) in sera of IM patients and normal controls by ELISA. It was found that Id 5488 is expressed both in IM patients and normal controls, and that Id 5488 expression is significantly higher in IM patients' sera; furthermore, in IM sera a statistically significant correlation between Id 5488 expression and anti-cytoskeleton and anti-smooth muscle autoantibodies was found. It is suggested that at least part of EBV-induced IgM autoantibodies appearing during IM are secreted by B lymphocytes programmed to the production of "natural antibodies" bearing Id 5488-like idiotopes. KEY WORDS: Epstein-Barr virus, monoclonal autoantibody, idiotype.
INTRODUCTION Epstein-Barr virus (EBV), the agent of infectious mononucleosis (IM), is proposed to be involved in the etio-pathogenesis of some diseases with autoimmune components, such as rheumatoid arthritis, systemic lupus erythematosus and Sjogren syndrome'. IM itself is characterized by a transient occurrence of autoantibodies (autoAbs) of the IgM class to several antigens, including, to mention only the most frequently found, those against smooth muscle and cytoskeleton antigens of epithelial cells, appearing in over 70-80% IM, therefore, represents an appropriate model for the study of the mechanism(s) by which EBV induces autoimmunity and, possibly, for investigations on the relationships of EBV to autoimmune diseases. The mechanism(s) responsible for the appearance of EBV-induced autoAbs are not exactly known, even though virus-induced polyclonal B cell activation seems to play the major role; in fact, it is generally accepted that, upon infection, EBV-transformed B lymphocytes are triggered to express their antibody
Correspondence to: Dr Carlo Garzelli, Dipartimento di Biomedicina, Sezione di Microbiologia, Via San Zeno, 39, 56100 Pisa. Italy.
repertoire, including autoAbs'-''. Mechanisms other than virus-induced polyclonal B cell activation, however, have been also proposed. They include: (a) autoimmunization with cell antigens released by EBVinduced tissue damage3*"; and (b) immunization with EBV-encoded nuclear antigen (EBNA- 1) bearing molecular homology with normal tissue proteins". One approach to the study of the mechanism(s) of EBV-induced autoimmunity has been the investigation of human monoclonal autoAbs (mAb) secreted by EBV-transformed B lymphocytes: it has been shown that a large part of EBV-transformed B lymphocytes from normal individuals or patients with autoimmune diseases produce monoclonal IgM autoAbs that, when studied in detail, turned out to be polyspecific or reactive with antigens in multiple o r g a n ~ ~ . ' ' , ' ~in; this context, we have recently described a human monoclonal autoAb, designated CAT- 182-6-3, produced by EBV-transformed B lymphocytes isolated from a patient in the acute phase of IM, reactive with both cytoskeleton filaments and smooth muscle, as well as with EBNA14, raising the question of the relevance of this autoAb in EBVinduced autoimmunity. Since the role of autoAbs in autoimmunity can be usefully approached through idiotype studies, in that it is known that in mice and humans certain idiotypes are closely associated with particular autoimmune dis171
172
C. GARZELLI ET AL.
MATERIALS AND METHODS
specificity, assumed as Id 5488-negative (or minimally expressed), in 100 p l of carbonate-bicarbonate buffer, pH 9.6. The bound mouse antibody was detected using peroxidase-labeled goat anti-mouse IgG (7-chain specific; Sigma, USA). The positive wells showing reactivity with HY 5488 mAb, but not with polyclonal human IgM were cloned using the limiting dilution method. One hybridoma producing monoclonal IgGl antibody, coded HY 3D5-05, was obtained and finally produced into ascites in Balbk mice.
Production and purification of human monoclonal antibody
ELISA assay f o r HY 5488 idiotope (Id 5488) in human sera
The procedure for the isolation of EBV-positive CAT1E2-6-3 cells has been reported in detail elsewhere14. In this study, in order to achieve higher antibody production in culture supernatant, 2x10' CAT-1E2-6-3 cells were fused with 2x10' ouabain-resistant P3.X63.AG8.653 mouse myeloma cells and human -mouse heterohybrids were selected in ouabain -containing HAT medium, as previously described''. A stable human-mouse heterohybrid, coded HY 5488, was isolated after 4 limiting dilution cloning procedures. This hybrid produced up to 80 pglml of monoclonal IgM in spent culture medium with the same antibody reactivity as parent CAT-1 E2-6-3 EBV-positive lymphoblastoid cells. HY 5488 mAb was then purified from culture supernatant using an immunoaffinity column, which was prepared by coupling the IgG fraction of goat anti-human IgM ( p chainspecific; Sigma Co., USA) to CNBr-activated Sepharose 4B (Pharmacia Fine Chemical, Sweden). The adsorbed antibody was eluted with 0.1 M glycineHC1, pH 2.5. The purity of HY 5488 mAb was tested by SDS-polyacrylamide gel electrophoresis (PAGE), and two only bands having the apparent molecular weights of p- and k-chains were detected by silver stain.
Immunolon I1 ELISA plates were coated with l o o p 1 of 1 5 0 0 dilution in carbonate-bicarbonate buffer, pH 9.6, of HY 3D5-05 ascites at 4°C overnight. After 3 washes in phosphate buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tw), l o o p 1 of human sera diluted 1:lOO in PBS-Tw were added to the wells in duplicate and incubated at room temperature for 1 hr. After 3 washes in PBS-Tw, the wells were incubated for 1 hr at r.t. with l o o p 1 of 1:lOOO dilution in PBSTw of peroxidase-labeled goat anti-human IgM ( p chain specific; Sigma). After 5 final washes, plates were developed with 0.04% H202and 0.04% o-phenylenediamine in phosphate-citrate buffer, pH 5.0. The optical density at 492 nm was read using a Multiscan Microplate Reader (Flow Labs). Wells containing serial 2-fold dilutions of a.p. HY 5488 mAb, ranging from 5 ng/ml to 10 pg/ml, served as standards.
Preparation of anti-idiotypic monoclonal antibody
ELISA assay f o r Epstein-Barr virus viral capsid antigen (VCA) and nuclear antigen ( E B N A )
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
~ r d e r s l ~and , ' ~ only a limited number of Ig families are involved in autoimmunity", we have prepared a murine mAb directed against an idiotypic determinant o f the human CAT-1E2-6-3 mAb; furthermore, using this anti-idiotypic mAb we have studied the expression of the corresponding idiotope on IgM in the sera of normal individuals and patients with IM and its relationship to EBV-induced autoantibodies.
Balb/c female mice were immunized by 3 weekly, intraperitoneal injections of 50, 25, and 25 pg, respectively, of affinity purified (a.p.) HY 5488 mAb and by a last intravenous injection of 2 5 p g of a.p. mAb. Three days later, splenic lymphocytes were fused with P3.X63.AG8.653 mouse myeloma cells using 50% polyethylene glycol at neutral pH. The fused cells were plated in 96-well tissue culture plates and incubated at 37°C. Supernatants from outgrowing hybrids were screened for anti-HY 5488 and antihuman IgM antibody by ELISA, in which Immunolon I1 plates (Dynatech Laboratories, USA) were coated with 1 p g of either a.p. HY 5488 mAb or purified polyclonal IgM (Cappel Lab., USA) with irrelevant
IM sera Sera were obtained from patients with the clinical and haematological characteristics of JM, in whom the diagnosis had been confirmed by a positive Monospot test and by the presence of IgM antibodies to EBV viral capsid antigen (VCA).
Microtiter 96-well plates, coated with VCA or EBNA antigen, purified via monoclonal antibodies, were purchased from Dupont (Billerica, MA, USA). For assays, wells were incubated at r.t. for 1 hr with 20Opl of sera diluted 1:lOO in PBS-Tw. After 3 washes in PBS-Tw, the wells were incubated for 45 min at r.t. with 2 0 0 p l of 1:lOOO dilution in PBSTw of biotin-labeled goat anti-human IgM ( p chain specific, Sigma). After 3 additional washes as before, the wells were incubated for a further 45 min at r.t. with 200 pl of a 1 :1000 dilution in PBS-Tw of peroxidase-labeled anti-biotin (Sigma). After 5 final washes, plates were developed as described above.
IDIOTYPE EXPRESSION ON EBV-INDUCED AUTOANTIBODY
standard immunofluorescence techniques (not shown).
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
ELISA assay for anti-smooth muscle and anti-cytoskeleton autoantibody
On the ground of previous reports indicating that in IM anti-smooth muscle autoantibodies are mainly directed against tubulin subunits6, the method 0-f Shelanski, Gaskin and Cantor'' for the purification of this molecule was followed, with some modifications. Briefly, a sample of tissue from human stomach, obtained from a surgical specimen, was washed in cold PBS, minced using scissors, suspended (approximately 1 g/ml) in 0.1 M Hepes buffer, pH 6.8, containing 1 mM EGTA, 1 mM GTP and protease inhibitors, and then homogenized by using a Polytron blender at 4°C. After centrifugation at 4°C for 1.5 hr at 40,00Og, the pellet was discarded and the supernatant was mixed with an equal volume of 8 M glycerol in buffer and the resultant mixture was stirred at 37°C for 30 min. After centrifugation at 100,000g for 1.5 hr at 22"C, the precipitate, containing partially purified tubulin, was resuspended in carbonate-bicarbonate buffer, pH 9.6. ELISA plates were coated at 4°C overnight with 1 pg in 100pl of the tubulin preparation. For anti-cytoskeleton autoAbs assay, confluent microcultures of Hep-2 cells were grown in 96-well plates, washed three times with PBS, and then fixed with 0.1% glutaraldehyde in PBS for 5 min at r.t. The cells were washed once, incubated for 5 min at r.t. in 1% bovine serum albumine (BSA) in PBS, and then washed twice more before storage in PBS at 4°C. For both assays. wells were washed three times with PBS-Tw, incubated at r.t. for 1 hr with 100 pl of sera diluted 1:lOO in PBS-Tw, and then developed as described above. In preliminary experiments, the results of the above reported ELISA assays were shown to correlate with d
1.200 c E
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x
,
1-
0
0.800 -
0.
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and reactivity of HY 5488
human mAb Approximately one year after EBV transformation, CAT- I E2-6-3 cells, producing few micrograms of human monoclonal IgM per ml of spent culture medium, were fused with ouabain-resistant mouse myeloma cells and the resulting human-mouse hybrids were selected in HAT medium supplemented with ouabain. After 4 cloning procedures by limiting dilution, a stable heterohybrid, chosen on the basis of high IgM production, was isolated and expanded in culture medium. This hybridoma, coded HY 5488, secreted up to 80 pg/ml of monoclonal human IgM in spent culture medium. HY 5488 mAb was then produced by expanding the heterohybrid in 75 cm2 tissue culture flasks; the culture supernatant was concentrated approximately 10-fold by ultrafiltration and finally purified to homogeneity by affinity chromatography. The affinity-purified HY 5488 mAb showed the same antibody reactivity as parent CAT-lE2-6-3derived mAbI4, in that it reacted with both cytoskeleton filaments of cultured human epithelial cells and smooth muscle of frozen sections of human stomach by immunofluorescence, as well as with EBVencoded nuclear antigen (EBNA) by ELISA. Preparation of monoclonal anti-idotypic antibody Anti-idiotypic antibody was prepared by fusing spleen cells from Balb/c mice, immunized with a.p. HY 5488 mAb, with P3.X63.AG8.653 mouse myeloma cells.
a
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RESULTS
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Figure 1 Specificity of HY 3D5-05 mAb for Id 5488. (Left), ELISA plate wells, coated with HY 3D5-05 mAb, received either serially diluted a.p. HY 5488 human mAb (circle) or the same concentrations of purified polyclonal IgM with irrelevant specificity, assumed as Id 5488-negative (triangle). (Right), E L S A plate wells, coated with either HY 3D5-05 ascites (circle) or P3.Xf~3.AG8.653 ascites (triangle), received serially diluted a.p. HY 5488
Autoimmunity Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/30/14 For personal use only.
174
C. GARZELLI ET AL.
One successful fusion (480 wells) yielded a total of six outgrowing hybridomas that reacted with HY 5488 mAb by ELISA. After further testing and cloning, one of these, designated HY 3D5-05, was found to react with a.p. HY 5488 mAb, but not with purified polyclonal human IgM, assumed as Id 5488-negative (or minimally expressed) on the basis of their irrelevant specificity. The specificity of binding of HY 3D5-05 mAb to Id 5488 is further shown in Figure 1 (left), where it can be seen that wells coated with HY 3D505 murine mAb bind a.p. HY 5488 human mAb, but not the Id 5488-negative, purified polyclonal human IgM. To test the formal possibility that HY 5488 mAb had an affinity for mouse ascites antigens, the binding of a.p. HY 5488 mAb to wells coated with either HY 3D5-05 ascites or aspecific P3.X63.AG8.653 ascites was compared; as shown in Figure 1 (right), HY 5488 mAb strongly bound to wells coated with HY 3D5-05 mAb, as expected, and in a much lower extent to the wells coated with the aspecific ascites. By western-blotting, the HY 3D5-05 murine mAb failed to react with either heavy or light chains of the a.p. HY 5488 mAb blotted from SDS-PAGE gel run in reducing conditions (not shown).
relates with the titers of EBV-induced IgM autoantibodies. This analysis was performed by plotting the serum concentration of Id 5488-bearing IgM against the optical reading values of anti-VCA, anti-EBNA, anti-cytoskeleton, and anti-smooth muscle IgM antibody, respectively. Anti-VCA and anti-EBNA IgM antibody assays employed commercially available purified antigens; anti-cytoskeleton IgM autoantibodies were assayed by an ELISA assay employing glutaraldehyde-fixed Hep-2 cells; anti-smooth muscle IgM were assayed by an ELISA assay employing a semi-purified preparation of tubulin, i.e., the autoantigen against which the IM anti-smooth muscle autoantibodies are directed6. The results of this analysis are reported in Figure 3; as can be seen, no statistically significant correlation was found between Id 5488 expression and anti-VCA or anti-EBNA IgM antibodies (upper, left and right), whereas a statistically significant correlation was found between the Id 5488 expression and anti-cytoskeleton (P=0.005) and anti-smooth muscle IgM autoantibodies (P=0.003) (lower, left and right, respectively).
Detection of Id 5488-bearing IgM in normal and infectious mononucleosis sera
By fusing EBV-transformed human B lymphocytes with mouse myeloma cells, we have prepared stable
Twenty-nine sera from normal individuals, with no significant anti-VCA IgM activity, and 31 sera from patients in the acute phase of IM, as assessed according to the criteria reported in the Materials and Methods section, were tested by ELISA for the expression of Id 5488-bearing IgM. The results are reported in Figure 2. As can be seen, Id 5488 was detectable in all sera tested, but considerable quantitative differences were found among individuals in both tested groups; in particular, in normal individuals, the concentration of Id 5488-bearing IgM ranged between 1 and 20 pglml, whereas in IM patients the Id 5488-bearing IgM ranged between 4 and 40 pglml. The differences between the means (6.9 pg/ml and 15.2 pglml, for normal and IM patients, respectively) were statistically highly significant (P
