Scand. J. Immunot. 36, 193-200, 1992
Enhancement of the Antibody Response to Protein Antigens by Speeifie IgG under Different Experimental Conditions E, J. WIERSMA Departmctit of Itnmutioiogy, Box 582. BMC, Uppsala. Sweden, and Dcpitrtmcnt of Medical and Physiological Chemistry. Box 575. BMC, Uppsala, Sweden
Wicrsma EJ, Enhancement of Ihe Antibody Response to Protein Antigens by Specific IgG under Ditrercnt Fxperimental Conditions, Scand J Immunol 1992:36:193 200 Specific stimulation of the antibody response to protein iintigcns by IgG antibodies was studied in vivo-The response to TNP-coup!ed keyhole limpet haemocyanin (KLH-TNP) was enhanced by a TNP-spcciftc IgG monoclonal antibody, 7B4, as measured both in ELISA (enzyme-linked immunosorbent assay) and by ELISPOT (enzyme-linked immunospot) method. Enhancement was seen when using both high and low doses of antigen and antibody. The antibody response to TNP-coupled bovine serum albumin (BSA-TNP), bul not to TNP-couplcd ovalbumin (OATNP), tetanus toxoid (TT-TNP) or diphtheria toxoid (DT-TNP). was also etficiently augmented by 7B4, Enhancement was seen when using both high and low coupling ratio of TNP to KLH, The fact that IgG-mediiited enhancement is seen under many ditVerent experimental conditions suggests that it is a physiologically relevant phenomenon. In order lo enhance the response to KLH-TNP and BSA-TNP, 7B4 had lo be injected while antigen was circulating in blood. This supports that igG-mediated stimulation acts by concentrating circulating antigen into lymphoid centres. Erik J. Wier.mut, Depariment of Immunology, Univer.iily of loroniu. .Vtfdical Science.s BuiUiing, Toronto. Ontario M5S lAiS. Canada
IgG antibodies can enhance or inhibit the humoral immune response against its specific antigen, so-called "feedback regulation". Enhancement by IgG is obtained for soluble protein antigens [1-9] whereas IgG in combination with protein antigens in adjuvant [4, 10, II] or heterologous erythroeytes [12-14] induces suppression. The different immunoregulatory effects are dependent upon the antigen used, since one monoclonal antibody (MoAb) may have opposite effects on the response to different antigens [8. 9]. It is not known what physical parameters of antigen determines the direction of immunoreguiation. Enhancement indueed by passively adtninistered IgG is thought to reflect ihe action of endogenous natural IgG antibodies in a normal primary response, or the action of antibodies produced in a primary response on the secondary response [15. 16], Previous data suggest that IgGmediated enhancement acts by increasing the uptake of antigen in lymphoid follicles, thereby
giving a more efficient presentation of antigen to B cells [2, 5], Probably both complement reeeptors, recognizing C3 fragments attached to the IgG-protein antigen complexes, as well as Fc receptors for IgG mediate the enhanced antigenie tiptake [5. 17]. In this study, the stochiometric conditions under which, and antigens to which, enhancement occurs were investigated. By mjecting antibody before and after antigen has been cleared from the circulation, an attempt was made to determine if the phenomenon acts by increasing antigen uptake, MATERIALS AND METHODS Aniigen.s. Antigens from the following sources were used: KLH (keyhole limpet haemocyanin) (Calbiochem-Bchring C , La Jolla, CA, USA): BSA and HEL (hen egg lyso/yme) (Sigma, St Louis. MO, USA): OA (Worthington, Freehold, NJ. USA): DT (diphtheria toxoid) (SSL Copenhagen, Denmark): TT (tetanus toxoid) (SBL, Stockholm, Sweden and Wellcome Bio-
193
194
E.J. Wiersma
tech. Beckenham, UK), 2,4,6-trinitrobenzene sulphonic acid ('TNP", Sigttta) was coupled to proteins in 0,28 M cacodylate buffer pH 6.9 for BSA, OA, DT and TT, atid 0,1 M NaHCOj buffer pH 8.3 for KLH. After different inctjbation times at room temperature (20 C)(KLII: 12 min, BSA: 65 min, OA: 40 min, DT: 46 h, TT: 48 h). the reaction was stopped by an excess glycyl-glycin (Merck, Darmstadt, Germany), followed by extensive dialysis against PBS, The number of incorporated TNP residues was determined spectrophotometrically according to Ref, 18, Indexes (e,g, BSA-TNPn) indicate number of TNP per protein moleeule, except for KLM, where it indicates number ofTNP residues per 100-kDa subunit. Antigens were stored at 4 C as sterile solutions. Antibodie.'i. TNP-specifie murine MoAb C4007B4 (7B4, lgG2a) and CI901B4 (IB4, lgG2b) have been described [7], Essentially pure monoclonal antibodies, as judged from SDS gel electrophoresis (Phast System, Phartnacia, Uppsala, Sweden), were obtained by affinity chromatography purification of cell culture supernatant on Protein A-Sepharose (Pharmacia) [19], Antisera against KLH, BSA, OA, DT and TT were obtained by hyperimmunizatton of mice. Normal mouse serum was a pool of scrum from ten nonimmunized miee. MoAb were stored at 4 C as .sterile solulions, whereas antisera were stored at —20 C in small aliquots. Animals and immunization. Male CBA/Ca miee were obtained from A-Lab (Stockholm, Sweden). Groups of at least four mice were injected with IgG antibody (or not, for the controls), followed after 1 2 h by TNPcoupled antigen. Sometimes TT or HEL was injected together with TNP-coupled antigen lo serve as speciftcity controls. Both MoAb and iintigen(s) were given intravenously in 100 /il PBS without adjuvant. Animals were bled from the tail 14 days after immunization. ;ind sera were tested individually in ELISA. Alternatively, mice were killed at day 4 to 7, and their spleens were individually assayed for antibody produclion by the ELISPOT method, ELISA. Determination of unil values of specific antibody per ml mouse sera was performed as described in a previous paper [17], Briefly, microtitre plalcs were coated with antigen in PBS. 0,05"ii NaN^ at a concentration of 15 ^(g.ml for TT and DT, and 50 /ig.ml tor other proteins. Next, plales were incubated wilh experimental serum samples and a dilution series of the respective standard iiyperimttiune aniiserutn. After this, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (H-hL. Bio-Rad. Richmond, CA. USA) was applied, followed by />-nitrophenyl substrate (Sigma), Determination of unit values was made by comparing absorbance values of standard and test samples by the EL1SA+ (Meddata Inc. New York, NY, USA) or the ASSAY-ZAP (Biosoft, Cambridge, UK) computer program. ELISPOT awflv, A moditied version |20] oT the ELISPOT assay [21] was used for determination of tlie number of splenic B cells secreting IgM or IgG specific for KLH or HEL. Briefly, microtitre plates were coated with antigen, as above. After incubation of 100 ;il spleen cells in each well for 3 h at 37 C and in 7"/,, CO:, "i" enzyme conjugate was added; either AP-conjugated goat anti-mouse IgM or AP-conjugated sheep antimouse IgG (Jackson Lab, Inc, West Grove. PA, USA)
was applied and incubated overnight at 4 C, Spots were developed by adding substrate solution, containing 5brotno-4-chloro-3-indolyl phosphate (Sigma), for about 1 h at room temperature. The number of spots was counted in a stereoscopic microscope, Cauihali.\m
oj ^-^ I-KLH-TNF
ami ^--I-BS.A-TNP
in
riio. KLH-TNPi: and BSA-TNPn were labelled with '-^1 using the lactoperoxidase method [22], and incorporated 0.06*? and 0,050 fiC\ ' " I / ' g protein, respectively. In vivo catabolism was investigated by injecting 20 fig of protein into mice as above, and bleedtng them individually at different time-points afterwards. Sera, in parallel with an aliquot of the protein solution used for injection, were precipitated and washed once in 10% (wt Vol) trichloroacetic acid (TCA), Radioactivity of pellets was determined in a y-counter, .V/c7fM7ic,s, The significance of the differences between experimcntiil groups were calculated by Student's Ntcst, /•values were obtained from means and SD of the iogm values of antibody concentration, or from means and login values of number of spot-forming eells,
RESULTS Effects of MoAb 7B4 on the response to different prolein antigens The immunoregulatory effect of the TNPspecific MoAb 7B4 on the response to five different TNP-coupied antigens was tested. Twenty micrograms of antigen was injected, either alone or after 50 /jg 7B4 (Table 1). The response to KLH-TNP and BSA-TNP was efficiently enhanced by 7B4, whereas the response to TT-TNP was slightly, not reprodueibly. enhanced by 7B4. No significant effect was seen on the antibody response to OA-TNP and DTTNP. Effect of different amounts of KLH-TNP and MoAb 7B4 It was investigated whether different amounts of protein antigen and of passive IgG antibody influeneed the direction or degree of tnodulation of the antibody response (Table II). In Experiments 1 and 2, MoAb 7B4 enhanced the response to both a low dose (5 /(g) and a high dose (80 ^g) of KLH-TNP12. Stimulation was most efficient when using 5 fig of KLH-TNPi:. Five hundred and fifty micrograms of 7B4 enhanced the response lo 20 /(g KLH-TNPi: to a similar degree, whereas 5 or 0,5 /jg of 7B4 did not give significant enhancement (Expt 3) In Expt 1, 12,5 /ig of 7B4 yielded less efficient enhancement of the response to 5/(gof KLH-TNPj; than 50 fig 7B4, Experiments using 10 or 30/(g of other TNPspecific IgG MoAb gave significant, but often
Enhancement of the Ab Response by IgG TABLE I, Effects of TNP-spcciflc MoAb 7B4 on ihe antibody response lo different TNP-coupled proteins Antibody response Immunizations 50/jg7B4 + 20/
Enhancement of the Antibody Response to Protein Antigens by Speeifie IgG under Different Experimental Conditions E, J. WIERSMA Departmctit of Itnmutioiogy, Box 582. BMC, Uppsala. Sweden, and Dcpitrtmcnt of Medical and Physiological Chemistry. Box 575. BMC, Uppsala, Sweden
Wicrsma EJ, Enhancement of Ihe Antibody Response to Protein Antigens by Specific IgG under Ditrercnt Fxperimental Conditions, Scand J Immunol 1992:36:193 200 Specific stimulation of the antibody response to protein iintigcns by IgG antibodies was studied in vivo-The response to TNP-coup!ed keyhole limpet haemocyanin (KLH-TNP) was enhanced by a TNP-spcciftc IgG monoclonal antibody, 7B4, as measured both in ELISA (enzyme-linked immunosorbent assay) and by ELISPOT (enzyme-linked immunospot) method. Enhancement was seen when using both high and low doses of antigen and antibody. The antibody response to TNP-coupled bovine serum albumin (BSA-TNP), bul not to TNP-couplcd ovalbumin (OATNP), tetanus toxoid (TT-TNP) or diphtheria toxoid (DT-TNP). was also etficiently augmented by 7B4, Enhancement was seen when using both high and low coupling ratio of TNP to KLH, The fact that IgG-mediiited enhancement is seen under many ditVerent experimental conditions suggests that it is a physiologically relevant phenomenon. In order lo enhance the response to KLH-TNP and BSA-TNP, 7B4 had lo be injected while antigen was circulating in blood. This supports that igG-mediated stimulation acts by concentrating circulating antigen into lymphoid centres. Erik J. Wier.mut, Depariment of Immunology, Univer.iily of loroniu. .Vtfdical Science.s BuiUiing, Toronto. Ontario M5S lAiS. Canada
IgG antibodies can enhance or inhibit the humoral immune response against its specific antigen, so-called "feedback regulation". Enhancement by IgG is obtained for soluble protein antigens [1-9] whereas IgG in combination with protein antigens in adjuvant [4, 10, II] or heterologous erythroeytes [12-14] induces suppression. The different immunoregulatory effects are dependent upon the antigen used, since one monoclonal antibody (MoAb) may have opposite effects on the response to different antigens [8. 9]. It is not known what physical parameters of antigen determines the direction of immunoreguiation. Enhancement indueed by passively adtninistered IgG is thought to reflect ihe action of endogenous natural IgG antibodies in a normal primary response, or the action of antibodies produced in a primary response on the secondary response [15. 16], Previous data suggest that IgGmediated enhancement acts by increasing the uptake of antigen in lymphoid follicles, thereby
giving a more efficient presentation of antigen to B cells [2, 5], Probably both complement reeeptors, recognizing C3 fragments attached to the IgG-protein antigen complexes, as well as Fc receptors for IgG mediate the enhanced antigenie tiptake [5. 17]. In this study, the stochiometric conditions under which, and antigens to which, enhancement occurs were investigated. By mjecting antibody before and after antigen has been cleared from the circulation, an attempt was made to determine if the phenomenon acts by increasing antigen uptake, MATERIALS AND METHODS Aniigen.s. Antigens from the following sources were used: KLH (keyhole limpet haemocyanin) (Calbiochem-Bchring C , La Jolla, CA, USA): BSA and HEL (hen egg lyso/yme) (Sigma, St Louis. MO, USA): OA (Worthington, Freehold, NJ. USA): DT (diphtheria toxoid) (SSL Copenhagen, Denmark): TT (tetanus toxoid) (SBL, Stockholm, Sweden and Wellcome Bio-
193
194
E.J. Wiersma
tech. Beckenham, UK), 2,4,6-trinitrobenzene sulphonic acid ('TNP", Sigttta) was coupled to proteins in 0,28 M cacodylate buffer pH 6.9 for BSA, OA, DT and TT, atid 0,1 M NaHCOj buffer pH 8.3 for KLH. After different inctjbation times at room temperature (20 C)(KLII: 12 min, BSA: 65 min, OA: 40 min, DT: 46 h, TT: 48 h). the reaction was stopped by an excess glycyl-glycin (Merck, Darmstadt, Germany), followed by extensive dialysis against PBS, The number of incorporated TNP residues was determined spectrophotometrically according to Ref, 18, Indexes (e,g, BSA-TNPn) indicate number of TNP per protein moleeule, except for KLM, where it indicates number ofTNP residues per 100-kDa subunit. Antigens were stored at 4 C as sterile solutions. Antibodie.'i. TNP-specifie murine MoAb C4007B4 (7B4, lgG2a) and CI901B4 (IB4, lgG2b) have been described [7], Essentially pure monoclonal antibodies, as judged from SDS gel electrophoresis (Phast System, Phartnacia, Uppsala, Sweden), were obtained by affinity chromatography purification of cell culture supernatant on Protein A-Sepharose (Pharmacia) [19], Antisera against KLH, BSA, OA, DT and TT were obtained by hyperimmunizatton of mice. Normal mouse serum was a pool of scrum from ten nonimmunized miee. MoAb were stored at 4 C as .sterile solulions, whereas antisera were stored at —20 C in small aliquots. Animals and immunization. Male CBA/Ca miee were obtained from A-Lab (Stockholm, Sweden). Groups of at least four mice were injected with IgG antibody (or not, for the controls), followed after 1 2 h by TNPcoupled antigen. Sometimes TT or HEL was injected together with TNP-coupled antigen lo serve as speciftcity controls. Both MoAb and iintigen(s) were given intravenously in 100 /il PBS without adjuvant. Animals were bled from the tail 14 days after immunization. ;ind sera were tested individually in ELISA. Alternatively, mice were killed at day 4 to 7, and their spleens were individually assayed for antibody produclion by the ELISPOT method, ELISA. Determination of unil values of specific antibody per ml mouse sera was performed as described in a previous paper [17], Briefly, microtitre plalcs were coated with antigen in PBS. 0,05"ii NaN^ at a concentration of 15 ^(g.ml for TT and DT, and 50 /ig.ml tor other proteins. Next, plales were incubated wilh experimental serum samples and a dilution series of the respective standard iiyperimttiune aniiserutn. After this, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (H-hL. Bio-Rad. Richmond, CA. USA) was applied, followed by />-nitrophenyl substrate (Sigma), Determination of unit values was made by comparing absorbance values of standard and test samples by the EL1SA+ (Meddata Inc. New York, NY, USA) or the ASSAY-ZAP (Biosoft, Cambridge, UK) computer program. ELISPOT awflv, A moditied version |20] oT the ELISPOT assay [21] was used for determination of tlie number of splenic B cells secreting IgM or IgG specific for KLH or HEL. Briefly, microtitre plates were coated with antigen, as above. After incubation of 100 ;il spleen cells in each well for 3 h at 37 C and in 7"/,, CO:, "i" enzyme conjugate was added; either AP-conjugated goat anti-mouse IgM or AP-conjugated sheep antimouse IgG (Jackson Lab, Inc, West Grove. PA, USA)
was applied and incubated overnight at 4 C, Spots were developed by adding substrate solution, containing 5brotno-4-chloro-3-indolyl phosphate (Sigma), for about 1 h at room temperature. The number of spots was counted in a stereoscopic microscope, Cauihali.\m
oj ^-^ I-KLH-TNF
ami ^--I-BS.A-TNP
in
riio. KLH-TNPi: and BSA-TNPn were labelled with '-^1 using the lactoperoxidase method [22], and incorporated 0.06*? and 0,050 fiC\ ' " I / ' g protein, respectively. In vivo catabolism was investigated by injecting 20 fig of protein into mice as above, and bleedtng them individually at different time-points afterwards. Sera, in parallel with an aliquot of the protein solution used for injection, were precipitated and washed once in 10% (wt Vol) trichloroacetic acid (TCA), Radioactivity of pellets was determined in a y-counter, .V/c7fM7ic,s, The significance of the differences between experimcntiil groups were calculated by Student's Ntcst, /•values were obtained from means and SD of the iogm values of antibody concentration, or from means and login values of number of spot-forming eells,
RESULTS Effects of MoAb 7B4 on the response to different prolein antigens The immunoregulatory effect of the TNPspecific MoAb 7B4 on the response to five different TNP-coupied antigens was tested. Twenty micrograms of antigen was injected, either alone or after 50 /jg 7B4 (Table 1). The response to KLH-TNP and BSA-TNP was efficiently enhanced by 7B4, whereas the response to TT-TNP was slightly, not reprodueibly. enhanced by 7B4. No significant effect was seen on the antibody response to OA-TNP and DTTNP. Effect of different amounts of KLH-TNP and MoAb 7B4 It was investigated whether different amounts of protein antigen and of passive IgG antibody influeneed the direction or degree of tnodulation of the antibody response (Table II). In Experiments 1 and 2, MoAb 7B4 enhanced the response to both a low dose (5 /(g) and a high dose (80 ^g) of KLH-TNP12. Stimulation was most efficient when using 5 fig of KLH-TNPi:. Five hundred and fifty micrograms of 7B4 enhanced the response lo 20 /(g KLH-TNPi: to a similar degree, whereas 5 or 0,5 /jg of 7B4 did not give significant enhancement (Expt 3) In Expt 1, 12,5 /ig of 7B4 yielded less efficient enhancement of the response to 5/(gof KLH-TNPj; than 50 fig 7B4, Experiments using 10 or 30/(g of other TNPspecific IgG MoAb gave significant, but often
Enhancement of the Ab Response by IgG TABLE I, Effects of TNP-spcciflc MoAb 7B4 on ihe antibody response lo different TNP-coupled proteins Antibody response Immunizations 50/jg7B4 + 20/
