Seand. J. Immunol. 35, 137-148. 1992
In Vitro Activation of Human T Lymphocytes by Haemophilus influenzae Type b Capsular Polysaccharides C, C, A, M. PEETERS*. A,-M, TENBERGEN-MEEKES, C, J. HEIJNEN. J, T. POOLMANt, B. J. M. ZEGERS & G. T, RIJKERS Department of Immunology. University Hospital for Children and Youth "Hct Wilhelmina Kinderziekenhuis", Utrecht, and t Unit for Bacterial Vaccine Development and Pathogenesis Research, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands
Peeters CCAM. Tcnbergen-Mcckcs A M , Heijiieii CJ, Pooltiian JT, Zegers BJM, Rijkers GT. In Vitro Activation of Huniiin T t.yiTiphoL'ylcs by Haemophilus mfluen:ae Typo h Capsular Polysacchiirides. Scitnd J Immtinol 1992:.15:l.l7-4'8 Polyribosilrihitolphosphatc (PRP), the capsular pnlysaccharidc frotn Haemophilus in/luenzae type b. is a T-cclMndepetidcnt type 2 antigen. Iti vilro culture of adult peripheral blood T cells with l5/(g,.ml PRP leads to induction of inlerleukin-2 receptor (I L-2R) expression on up to \0'V., ofT cells. These cells arc CD4' and curry ihe a/J T-cell receptor. PRP, at conccntrattoiis above 1 5/(g till, can also induce in vitro pruiifora tion of bolh adult and neonatan cells. We conclude (hat PRP acis as a human T-cell mitogen. The in vjlro prolilcrative response as well as IL-2R expressioti was studied in T cells derived from adults after vaceindtion with native PRP. with PRP conjugated loa carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP rcqutred for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activatton. C C. A. M. Peeters. Unit for Bacterial Vaceiru' Detelopmcnt and Pathogenesis Riwearch. National Itisiitute oJ Public Health and Emironmetual Protection. P.O. Box I. 3720 BA Billhoien. The Netherlands
The capsular polysaccharides of encapsulated bacteria such as pneumococci and Haemophilu.s influenzae type h are characterized as T-ccllindependent type 2 (TI-2) antigens [1, 2]. This implies that T cells are not required for the induction of an antibody response to these antigens, but that the magnitude of the antibody response may be significantly influenced by T cells. Extensive studies in mice on the regulatory role of T cells have shown that the magnitude of an anti-polysaccharide response can be influenced by amplifier T cells (Ta), cells whieh function primarily by expanding clones of antigen-stimulated B cells. On the other hand, suppressor T ceils (Ts). that down-regulate the anti• Present address; Unit for Bacterial Vaceine Development and Pathogenesis Research, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
polysaeehaHde response, have been documented [i. 3 7]. In man, the antibody response to polysaccharide antigens shows characteristics similar to that in mice {reviewed in Ref. 8). The TI-2 nature of a limited number of polysaccharide antigens has been described [9, 10], and, more recently, the regulatory role of T cells in the human in vitro antibody response to pneumococcal poiysaccharides type 2 and type 4 has been docutnented. Conjugation o{ bacterial polysaccharides to carrier proteins changes the nature of an ensuing anti-polysaccharide antibody response from a TI-2 into that of a T-cell-dependent (TD) response. In contrast to native polysaccharides, polysaccharide-protcin conjugates are able to prime for booster responses and are able to induce an antibody response in children younger than two years of age [11-13]. The cellular immunological mechanisms 137
138
C.C. A. M. Pi'ctcrs et al.
underlying the improved immunogenicity ofconjugated versus native polysaccharides are incompletely understood. Recently, it has been demonstrated that T cells are required for the induction of an immune response to a Haemophilus influcn2ac type h oligosaccharidc-protcin conjugate vaccine (11], Whether the improved immunogenicity of polysaccharide-protein conjugates is due to the activation of carrier-specilic T cells, or whether polysaccharide-specific T cells are involved, is unclear. The existence of T celis which can be activated by polysaccharide antigens is controversial. Two papers froin the mid 1970s suggest that the capsular polysaccharide from Haemophilus influenzae type h (poly 3/f-D-ribose (1-^1) ribitol-5-phosphate (PRP)) can induce in vitro T-cell proliferation [14, 15], More recently, il has been reported that vaccination with polysaccharide vaccines may lead to in vivo T-cel! activation [16-19], In this study we have investigated whether vaccination with native or conjugated PRP leads to in vivo T-cell activation or may prime T ceils to become responsive to restimulation by PRP in vitro. To that end. the ability of PRP to induce interleukin-2 receptor (iL-2R) expression and Tcell proliferation was studied in cells from adults vaccinated with either native PRP or the Haemophilus influenzae type h oligosaccharide mutant diphtheria toxin conjugate vaccine (HbOC). Our data show that in vitro activation with PRP can induce proliferation of T cells from non-vaccinated adults as well as cord biood T cells. Vaccination with cither native PRP or the otigosaccharidc-protein conjugate increased the in vitro PRP-induced T-cell proliferation.
MATERIALS AND METHODS s. Kivc dirferenl batches of highly purilied PRP (viiccitu; qiKilily) were oblaincd from Dr D. Schtilz, Institul Mcrietix, Marcy LTtoile, F-'rancL', and from I>rP. Anderson, Department of Pediatrics, Scliool of Medicine, University of Rochester. Rocheslcr, New York. USA. Lipopolysaccharide (LPS) from Hiienuipliilu\ infiuenzac type h was obtained from Dr D. Schulz. Otiler membrane protcm P2 (OMP) of Ihe unencapsulated Haemcphilus influenziie strain DI was kindly donated by Dr L. van Alphen (Department of Medical Microbiology. Universily of Amsterdam, Amsterdam, The Netherlands). Diphtheri:i loxoid (DT; 150 limit lloctulalion units/ml) wa.s obtained from the National Instituli^ nf Public Health and Environmental Protection, Bilthoven, The Netherlands. All antigens were
dissolved in 0.9".l, NaCI and kepi al 4 C under sterile conditions. The ciidotoxin content of PRP was determined by the Limulus amoehocyte assay. Itnmunizuiion.s. Eighl healthy adult volunleers. aged 20-50 years (male:female ratio y:\) were vaccinated intramuscularly with a ().5-ml dose of the HbOC conjugate vaccine (Hibliter: Praxis Biologies. Rochester, N.Y.. USA}, containing 10 /jg Hacfuiphilu.s infiuenzav type h oligosaccharide and approximately 25 /ig mutant diphtheria toxin (CRM197). Four healthy adult volunteers were vaccinated intratnuscularly wilh a 0.5ml dose of PRP (CAPSA; Praxis Biologies) containing 25 /(g of the Haemiiphilu.s influenzae type h polysaccharide. Another tour healthy adull volunteers were vaccinated intramuscularly witli a I.O-ml doscof a combination vaccine consisting of diphtheria toxoid. tetanus toxoid, and poliotnyelitis (DTP; National Institute of Public Health and Environmental Protection). All vaccinees were routinely vaccinated with DTP during childhood (2()J. Blood was obtained from the vaceinees before and al various titnes after vaccination. Cord blood was obtained from neonates born to healthy mothers after uneventful pregnancies. Cell i.whilion ami culture. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by density gradient ccntrifugation on Ficoll Isopaque {p= 1.077 g.cm'; Pharmaeia. Uppsala, Sweden) at 1000 g for 20 min. Celis were washed twice with MEM-Tris (Tris-buffered minimal essential medium; Gibco, Grand Island, N.Y., USA). T cells were separated from PBMC by rosetting with AET (2aminocthylisothiouroniumbromide: Sigma Chemieal Co.. St. Louis. Mo., USA)-trealed sheep erythrocytes [21]. Non T cells were suspended in MEM. iO"'.. fetal calf serum (PCS) and irradiated in a Gammacell 1000 gamma irradlalor (Isomedix. Parsipanny. N.J.. USA) to receive a total dose of 3000 rad. PBMC or T cells reconstituted with irradiated autologous non T cells were cultured at a density of I x 10*/ mi in llat-bottom 96-welt polystyrene niierolitre plates (Nunc, Glostrup. Denmark) in medium consisting of RPMI-1640. supplemented with 200 /ig/ml glutamine. penicillin (100 lU/ml), streptomycin (100 /ig/ml), 0.05 mM 2-mercaptoethanol and i5"ii heat-inactivated pooled human A B serum. Cells were cultured at 37 Cin a humidified atmosphere of S'Hi CO;: in air for a period of six days. During the last 16 h of culture, individual wells were pulsed with 1 /iCi (37 kBq) ['H]thyiiiidine (specific activity 75 gBq mmol; Amersham International, Amersham. UK). Cells were harvested on a cell harvester (Skatron SA. Lier, Norway) and ['H]thymidine incorporation (in cpm) was measured in an LKB liquid scintillation counter. Results are presented as arithmetic means ( ± standard error) of four- to sixfold replicate cultures. Stimulation indices were determined by calculating ihe ratio of ["H|lhymidine incorporation in the presence of antigen and ['HJlliymidine incorporation in the absence of antigen. IL'l and IL-2 re.spon.\h-ene.s.s. T cells were cultured wilh antigen and irradiated autologous non T cells or variable amounts (0.1 10 U/ml) of human rlL-l/( (Dr A. Shaw, Glaxo Institute of Molecular Biology, Geneva, Switzerland). The lL-1 activity of the preparation was determined in a DI0.G4.1 proliferation assay [22] in the presence of saturating concentrations of lL-2
Activation of T Lymphocytes hy 11. infiucnzae Polysitccharide.s and IL-4: 0,05 U IL-l/|ml resulted in oplimal proliieration of D1O.G4,1 cells (stimulation index 25), IL-2 responsiveness was determined by culturing T cells, reconstituted with irradiLited autologous non T cells, with antigen as described above. After 72 h of culture, human rIL-2 (Boehringer Mannheim. Mannheim, Germany) was added to the cultures in a final eoncentration of 5 U/ml, After another 4S h of culture, individual wells were pulsed wilh I'Hlthymidine and processed as described above. Flow cyionu'iry. Cultured cells were stained for twocolour KACS analysis hy sequential incubation of 0,5 X 10*' PBMC with saturating amounts of primary antibody followed by an incubation with fluoresccin isothiocyanate (FITC')-conjugated goat anti-mouse Ig. and phycoerythrin (PE)-conjugated secondary imtibody. Each incubation was carried out in MEM, 1"'> bovine serum albumin (BSA) 0.05";) Na-azide for 30 tnin at 4 C and was followed by three washings. Stained eells were run on a F'ACS Analyzer (Beeton-Dickinson. Mountain View, Calif,. USA), equipped with a 250 mW Argon laser (EACSIight). Ten thousand events were stored per sample in list mode and analysed by using Consort 30 (Becton-Diekinson) software. Unconjugated or PE-conjugated anti-lL-2 receptor (CD25), Leu 4 (CD3). PF-conjugated Leu 3 (CD4) and PE-conjugated Leu 2 (CDS) were obtained from Becton-Dickinson: OKT! I (CD2) was from Ortho (Raritan, N.J.. USA). The anti-3;/( T-cell receptor antibody WT3I was kindly provided by Dr W. Tax (Department of Nephrology. University of Nijmegen, The Netherlands). Determination of anii-PRP ai}lihinlie\. Serum antiPRP antibodies were determined by enzyme-linked immunosorbent assay (ELiSA), Ninety-six-well highactivated immunoassay microplates (Tiiertek. Klow Laboratories Inc.. Irvine, UK) were coated with PRPby an incubation of 100 /il of tyramine-coupled PRP [23] (5- 10/ig, ml) in 0.9" n NaCl overnight at 37 C [24]. After washing with PBS, 0.05"/,. (wt vo!) Tween-20. plates were incubated with serial dilutions of the serum samples and a standard serum for 2 h at .^7 C. Plates were washed again and subsequently incubated with 100 ^1 goat anti-human IgM, IgG, or polyvalent Ig peroxidase(PO)-Iabelled conjugate (Tago. Burlingame. Calif,. USA) for 1 h at .17 C. After washing with PBS,
0,05"/,, Tween-20. 100 /il of the PO substrate (042 niM 3,3'. 5.5'tetramelhylben/idiiie (Sigma), 0.003'!.. H:O.in 0,11 M Na acetate bull'er, pH 5.5) was added to each weli. The reaction was stopped with 100 /il 2 N H1SO4, Optical density was measured on a Titertek FLISA reader at 450 nm. An anti-PRP hyperimmune serum (containing 32,5 /ig specific antibody ml [25]) was used as a standard. The standard was assigned arbitrarily to a level of lOO",. of IgM anti-PRP and 100"-,, IgG antiPRP, Antibody concentrations were calculated by determining the ratio of dilutions of both reference and sample of parallel running lines and arc expressed as a percentage of the reference serum. S/iiii.\iics. Antibody concentrations were log converted before statistical analysis. Results are expressed as geometric means " SE (aniilog of SEM of the logs). Significance of ditlerenees was tested by one-way or two-way variance analysis followed by a Student Newman-Keul's test with an overall significance level (a) of 0,05. Changes in magnitude and dose respon.siveness in f-cell proliferation following vaccination were assessed by analysis of variance with repeated measurements.
RESULTS
PRP-incluced in rifro T-cell proliferation In a first series of experiments. T cells, obtained from noti-vaccinated adults, were reconstituted with irradiated non T cells and cultured in vitro with P R P . T cells were reconstituted with noti T cells in a ratio as present initially in unseparated P B M C . In vitro culture with P R P (batch 103a. Table I) induced a significant level of T-cell proliferation (/''>mt^iinc iticorporation of cuUures stimulated wilh pokeweed miiogen (50 /ig/ml) was 47,293±198i cpm for aduh T cells and 35,128±2597 cpm for neonatal T cells.
proliferation of eord blood T eells (F'ig. 1). Both dose dependency and magnitude of [he response were similar to those observed with adult T eells. In vitro activation with 0.075-25 ftglm] DT did not result in significant proliferation of eord blood T cells (stimulation indices
In Vitro Activation of Human T Lymphocytes by Haemophilus influenzae Type b Capsular Polysaccharides C, C, A, M. PEETERS*. A,-M, TENBERGEN-MEEKES, C, J. HEIJNEN. J, T. POOLMANt, B. J. M. ZEGERS & G. T, RIJKERS Department of Immunology. University Hospital for Children and Youth "Hct Wilhelmina Kinderziekenhuis", Utrecht, and t Unit for Bacterial Vaccine Development and Pathogenesis Research, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands
Peeters CCAM. Tcnbergen-Mcckcs A M , Heijiieii CJ, Pooltiian JT, Zegers BJM, Rijkers GT. In Vitro Activation of Huniiin T t.yiTiphoL'ylcs by Haemophilus mfluen:ae Typo h Capsular Polysacchiirides. Scitnd J Immtinol 1992:.15:l.l7-4'8 Polyribosilrihitolphosphatc (PRP), the capsular pnlysaccharidc frotn Haemophilus in/luenzae type b. is a T-cclMndepetidcnt type 2 antigen. Iti vilro culture of adult peripheral blood T cells with l5/(g,.ml PRP leads to induction of inlerleukin-2 receptor (I L-2R) expression on up to \0'V., ofT cells. These cells arc CD4' and curry ihe a/J T-cell receptor. PRP, at conccntrattoiis above 1 5/(g till, can also induce in vitro pruiifora tion of bolh adult and neonatan cells. We conclude (hat PRP acis as a human T-cell mitogen. The in vjlro prolilcrative response as well as IL-2R expressioti was studied in T cells derived from adults after vaceindtion with native PRP. with PRP conjugated loa carrier protein, or with diphtheria toxoid. Vaccination with conjugated PRP decreased the doses of PRP rcqutred for in vitro induction of IL-2R expression and T-cell proliferation. This indicates that vaccination with PRP conjugated to a carrier protein improves the in vitro T-cell response to PRP activatton. C C. A. M. Peeters. Unit for Bacterial Vaceiru' Detelopmcnt and Pathogenesis Riwearch. National Itisiitute oJ Public Health and Emironmetual Protection. P.O. Box I. 3720 BA Billhoien. The Netherlands
The capsular polysaccharides of encapsulated bacteria such as pneumococci and Haemophilu.s influenzae type h are characterized as T-ccllindependent type 2 (TI-2) antigens [1, 2]. This implies that T cells are not required for the induction of an antibody response to these antigens, but that the magnitude of the antibody response may be significantly influenced by T cells. Extensive studies in mice on the regulatory role of T cells have shown that the magnitude of an anti-polysaccharide response can be influenced by amplifier T cells (Ta), cells whieh function primarily by expanding clones of antigen-stimulated B cells. On the other hand, suppressor T ceils (Ts). that down-regulate the anti• Present address; Unit for Bacterial Vaceine Development and Pathogenesis Research, National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
polysaeehaHde response, have been documented [i. 3 7]. In man, the antibody response to polysaccharide antigens shows characteristics similar to that in mice {reviewed in Ref. 8). The TI-2 nature of a limited number of polysaccharide antigens has been described [9, 10], and, more recently, the regulatory role of T cells in the human in vitro antibody response to pneumococcal poiysaccharides type 2 and type 4 has been docutnented. Conjugation o{ bacterial polysaccharides to carrier proteins changes the nature of an ensuing anti-polysaccharide antibody response from a TI-2 into that of a T-cell-dependent (TD) response. In contrast to native polysaccharides, polysaccharide-protcin conjugates are able to prime for booster responses and are able to induce an antibody response in children younger than two years of age [11-13]. The cellular immunological mechanisms 137
138
C.C. A. M. Pi'ctcrs et al.
underlying the improved immunogenicity ofconjugated versus native polysaccharides are incompletely understood. Recently, it has been demonstrated that T cells are required for the induction of an immune response to a Haemophilus influcn2ac type h oligosaccharidc-protcin conjugate vaccine (11], Whether the improved immunogenicity of polysaccharide-protein conjugates is due to the activation of carrier-specilic T cells, or whether polysaccharide-specific T cells are involved, is unclear. The existence of T celis which can be activated by polysaccharide antigens is controversial. Two papers froin the mid 1970s suggest that the capsular polysaccharide from Haemophilus influenzae type h (poly 3/f-D-ribose (1-^1) ribitol-5-phosphate (PRP)) can induce in vitro T-cell proliferation [14, 15], More recently, il has been reported that vaccination with polysaccharide vaccines may lead to in vivo T-cel! activation [16-19], In this study we have investigated whether vaccination with native or conjugated PRP leads to in vivo T-cell activation or may prime T ceils to become responsive to restimulation by PRP in vitro. To that end. the ability of PRP to induce interleukin-2 receptor (iL-2R) expression and Tcell proliferation was studied in cells from adults vaccinated with either native PRP or the Haemophilus influenzae type h oligosaccharide mutant diphtheria toxin conjugate vaccine (HbOC). Our data show that in vitro activation with PRP can induce proliferation of T cells from non-vaccinated adults as well as cord biood T cells. Vaccination with cither native PRP or the otigosaccharidc-protein conjugate increased the in vitro PRP-induced T-cell proliferation.
MATERIALS AND METHODS s. Kivc dirferenl batches of highly purilied PRP (viiccitu; qiKilily) were oblaincd from Dr D. Schtilz, Institul Mcrietix, Marcy LTtoile, F-'rancL', and from I>rP. Anderson, Department of Pediatrics, Scliool of Medicine, University of Rochester. Rocheslcr, New York. USA. Lipopolysaccharide (LPS) from Hiienuipliilu\ infiuenzac type h was obtained from Dr D. Schulz. Otiler membrane protcm P2 (OMP) of Ihe unencapsulated Haemcphilus influenziie strain DI was kindly donated by Dr L. van Alphen (Department of Medical Microbiology. Universily of Amsterdam, Amsterdam, The Netherlands). Diphtheri:i loxoid (DT; 150 limit lloctulalion units/ml) wa.s obtained from the National Instituli^ nf Public Health and Environmental Protection, Bilthoven, The Netherlands. All antigens were
dissolved in 0.9".l, NaCI and kepi al 4 C under sterile conditions. The ciidotoxin content of PRP was determined by the Limulus amoehocyte assay. Itnmunizuiion.s. Eighl healthy adult volunleers. aged 20-50 years (male:female ratio y:\) were vaccinated intramuscularly with a ().5-ml dose of the HbOC conjugate vaccine (Hibliter: Praxis Biologies. Rochester, N.Y.. USA}, containing 10 /jg Hacfuiphilu.s infiuenzav type h oligosaccharide and approximately 25 /ig mutant diphtheria toxin (CRM197). Four healthy adult volunteers were vaccinated intratnuscularly wilh a 0.5ml dose of PRP (CAPSA; Praxis Biologies) containing 25 /(g of the Haemiiphilu.s influenzae type h polysaccharide. Another tour healthy adull volunteers were vaccinated intramuscularly witli a I.O-ml doscof a combination vaccine consisting of diphtheria toxoid. tetanus toxoid, and poliotnyelitis (DTP; National Institute of Public Health and Environmental Protection). All vaccinees were routinely vaccinated with DTP during childhood (2()J. Blood was obtained from the vaceinees before and al various titnes after vaccination. Cord blood was obtained from neonates born to healthy mothers after uneventful pregnancies. Cell i.whilion ami culture. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by density gradient ccntrifugation on Ficoll Isopaque {p= 1.077 g.cm'; Pharmaeia. Uppsala, Sweden) at 1000 g for 20 min. Celis were washed twice with MEM-Tris (Tris-buffered minimal essential medium; Gibco, Grand Island, N.Y., USA). T cells were separated from PBMC by rosetting with AET (2aminocthylisothiouroniumbromide: Sigma Chemieal Co.. St. Louis. Mo., USA)-trealed sheep erythrocytes [21]. Non T cells were suspended in MEM. iO"'.. fetal calf serum (PCS) and irradiated in a Gammacell 1000 gamma irradlalor (Isomedix. Parsipanny. N.J.. USA) to receive a total dose of 3000 rad. PBMC or T cells reconstituted with irradiated autologous non T cells were cultured at a density of I x 10*/ mi in llat-bottom 96-welt polystyrene niierolitre plates (Nunc, Glostrup. Denmark) in medium consisting of RPMI-1640. supplemented with 200 /ig/ml glutamine. penicillin (100 lU/ml), streptomycin (100 /ig/ml), 0.05 mM 2-mercaptoethanol and i5"ii heat-inactivated pooled human A B serum. Cells were cultured at 37 Cin a humidified atmosphere of S'Hi CO;: in air for a period of six days. During the last 16 h of culture, individual wells were pulsed with 1 /iCi (37 kBq) ['H]thyiiiidine (specific activity 75 gBq mmol; Amersham International, Amersham. UK). Cells were harvested on a cell harvester (Skatron SA. Lier, Norway) and ['H]thymidine incorporation (in cpm) was measured in an LKB liquid scintillation counter. Results are presented as arithmetic means ( ± standard error) of four- to sixfold replicate cultures. Stimulation indices were determined by calculating ihe ratio of ["H|lhymidine incorporation in the presence of antigen and ['HJlliymidine incorporation in the absence of antigen. IL'l and IL-2 re.spon.\h-ene.s.s. T cells were cultured wilh antigen and irradiated autologous non T cells or variable amounts (0.1 10 U/ml) of human rlL-l/( (Dr A. Shaw, Glaxo Institute of Molecular Biology, Geneva, Switzerland). The lL-1 activity of the preparation was determined in a DI0.G4.1 proliferation assay [22] in the presence of saturating concentrations of lL-2
Activation of T Lymphocytes hy 11. infiucnzae Polysitccharide.s and IL-4: 0,05 U IL-l/|ml resulted in oplimal proliieration of D1O.G4,1 cells (stimulation index 25), IL-2 responsiveness was determined by culturing T cells, reconstituted with irradiLited autologous non T cells, with antigen as described above. After 72 h of culture, human rIL-2 (Boehringer Mannheim. Mannheim, Germany) was added to the cultures in a final eoncentration of 5 U/ml, After another 4S h of culture, individual wells were pulsed wilh I'Hlthymidine and processed as described above. Flow cyionu'iry. Cultured cells were stained for twocolour KACS analysis hy sequential incubation of 0,5 X 10*' PBMC with saturating amounts of primary antibody followed by an incubation with fluoresccin isothiocyanate (FITC')-conjugated goat anti-mouse Ig. and phycoerythrin (PE)-conjugated secondary imtibody. Each incubation was carried out in MEM, 1"'> bovine serum albumin (BSA) 0.05";) Na-azide for 30 tnin at 4 C and was followed by three washings. Stained eells were run on a F'ACS Analyzer (Beeton-Dickinson. Mountain View, Calif,. USA), equipped with a 250 mW Argon laser (EACSIight). Ten thousand events were stored per sample in list mode and analysed by using Consort 30 (Becton-Diekinson) software. Unconjugated or PE-conjugated anti-lL-2 receptor (CD25), Leu 4 (CD3). PF-conjugated Leu 3 (CD4) and PE-conjugated Leu 2 (CDS) were obtained from Becton-Dickinson: OKT! I (CD2) was from Ortho (Raritan, N.J.. USA). The anti-3;/( T-cell receptor antibody WT3I was kindly provided by Dr W. Tax (Department of Nephrology. University of Nijmegen, The Netherlands). Determination of anii-PRP ai}lihinlie\. Serum antiPRP antibodies were determined by enzyme-linked immunosorbent assay (ELiSA), Ninety-six-well highactivated immunoassay microplates (Tiiertek. Klow Laboratories Inc.. Irvine, UK) were coated with PRPby an incubation of 100 /il of tyramine-coupled PRP [23] (5- 10/ig, ml) in 0.9" n NaCl overnight at 37 C [24]. After washing with PBS, 0.05"/,. (wt vo!) Tween-20. plates were incubated with serial dilutions of the serum samples and a standard serum for 2 h at .^7 C. Plates were washed again and subsequently incubated with 100 ^1 goat anti-human IgM, IgG, or polyvalent Ig peroxidase(PO)-Iabelled conjugate (Tago. Burlingame. Calif,. USA) for 1 h at .17 C. After washing with PBS,
0,05"/,, Tween-20. 100 /il of the PO substrate (042 niM 3,3'. 5.5'tetramelhylben/idiiie (Sigma), 0.003'!.. H:O.in 0,11 M Na acetate bull'er, pH 5.5) was added to each weli. The reaction was stopped with 100 /il 2 N H1SO4, Optical density was measured on a Titertek FLISA reader at 450 nm. An anti-PRP hyperimmune serum (containing 32,5 /ig specific antibody ml [25]) was used as a standard. The standard was assigned arbitrarily to a level of lOO",. of IgM anti-PRP and 100"-,, IgG antiPRP, Antibody concentrations were calculated by determining the ratio of dilutions of both reference and sample of parallel running lines and arc expressed as a percentage of the reference serum. S/iiii.\iics. Antibody concentrations were log converted before statistical analysis. Results are expressed as geometric means " SE (aniilog of SEM of the logs). Significance of ditlerenees was tested by one-way or two-way variance analysis followed by a Student Newman-Keul's test with an overall significance level (a) of 0,05. Changes in magnitude and dose respon.siveness in f-cell proliferation following vaccination were assessed by analysis of variance with repeated measurements.
RESULTS
PRP-incluced in rifro T-cell proliferation In a first series of experiments. T cells, obtained from noti-vaccinated adults, were reconstituted with irradiated non T cells and cultured in vitro with P R P . T cells were reconstituted with noti T cells in a ratio as present initially in unseparated P B M C . In vitro culture with P R P (batch 103a. Table I) induced a significant level of T-cell proliferation (/''>mt^iinc iticorporation of cuUures stimulated wilh pokeweed miiogen (50 /ig/ml) was 47,293±198i cpm for aduh T cells and 35,128±2597 cpm for neonatal T cells.
proliferation of eord blood T eells (F'ig. 1). Both dose dependency and magnitude of [he response were similar to those observed with adult T eells. In vitro activation with 0.075-25 ftglm] DT did not result in significant proliferation of eord blood T cells (stimulation indices
