Isolation of a human T cell line specific for a streptococcal cell surface antigen

FEMS MicrobiologyImmunology76 (1991) 177-184 © 1991 Federation of European MicrobiologicalSocieties 0920-8534/91/$03.50 ADONIS 092085349100074D

177

FEMSIM 00163

Isolation of a human T cell line specific for a streptococcal cell surface antigen R . H . Brookes, L.S. Rayfield, L.A. B e r g m e i e r a n d P.S. S h e p h e r d Department of lmmunology, Medical School, UMDS, Guy's Campus, London, U.K.

Received 16 January 1991 Accepted 22 February 1991

Key words: Streptococcus mutans; Streptoccocal antigen I / I I ; Dental caries; Human T cell line

1. SUMMARY

2. I N T R O D U C T I O N

A streptococcal cell surface antigen of M r 185000 ( S A I / I I ) expressed by Streptococcus rnutans has previously been well characterised. A T cell line specific for native S A I / I I has been isolated from peripheral blood mononuclear cells (PBMC) of a naturally sensitised normal individual. This line has been maintained in culture for several months and was shown to be highly specific, not only for different preparations of native antigen but also for recombinant S A I / I I protein. It did not respond to a homologous antigen SpaA (M r 210000), extracted from Strep. sobrinus. The phenotype of the line was CD3 + CD4 + C D 8 - TcRafl +. HLA typing and inhibition studies showed that the response was restricted by both D R and DP encoded class II.

Members of the mutans Streptococci group have been implicated in the initiation and development of dental caries [1,2]. Of this group, the organisms most commonly associated with dental plaque in humans are Strep. mutans (serotype c) and Strep. sobrinus (formerly Strep. mutans serotype d). Investigations into mechanisms of immunity against caries have, therefore, focused on these bacteria. Several cell surface glycoproteins have been isolated from Strep. mutans [3], of these a glycoprotein of M r 185000 has been extensively characterised and been designated S A I / I I [4]. This immunodominant antigen has also been isolated by other groups and variously referred to as antigen B [5], PI [6], SpaP [7] and PAc [8]. S A I / I I is expressed abundantly on the cell surface of Strep. mutans [9,10] and is thought to act as an adhesin [11] and as a virulence factor [12]. A similar protein of M r 210000, named SpaA [13] (also known as PAg [14]) which may have the same function as S A I / I I and which shows serological cross reactiv-

Correspondence to: R.H. Brookes, Department of Immunology, 3rd Floor, Medical School, UMDS, Guy's Campus, London Bridge SE1 9RT, U.K.

178 ity [15] has also been isolated from cultures of Strep. sobrinus. Recently the complete D N A sequence for the gene encoding S A I / I I has been determined for Strep. mutans strains NG5 [16], and MT8148 [17], these have been called spaP and pac, respectively. The gene encoding SpaA (PAg), from Strep. sobrinus strain MT3791 [17], and known as pag, has also been sequenced. Comparison of the nucleotide sequences between the pac and pag genes has revealed a significant homology (62%) in their middle regions [17]. In most, if not all humans, IgG antibodies to S A I / I I can be readily detected in sera and T cells made to proliferate in vitro on stimulation with antigen [18-22]. As a first step in identifying major T cell epitopes on the molecule we have isolated and characterised a human T cell line specific for SAI/II.

3. MATERIALS A N D M E T H O D S 3.1. Tissue culture medium The standard medium used was RPMI 1640 (074-08100N, Gibco Ltd., U.K.) supplemented with 2 m g / m l sodium bicarbonate (10247, BDH, U.K.), 1 mM sodium pyruvate (16-820-49, Flow Labs, Scotland), 100 units/ml of penicillin and 100 t t g / m l streptomycin (043-05070H, Gibco), and 2.4 m g / m l Hepes (44285, BDH; p H 7.3). This was filter sterilised (0.22 btm) before use. All cultures were performed at 37°C in a humidified, 5% CO 2 atmosphere. 3.2. Antigens Native S A I / I I was prepared from either the supernatant or the cellular fractions of live cultures of Strep. mutans (Guy's strain) as described previously [3,9]. Supernatant proteins (each batch prefixed F) were extracted by ammonium sulphate precipitation, while cellular proteins (prefixed US) were extracted by solubilisation in urea. Both preparations were then further purified by ion exchange chromatography and gel filtration [3,4,9]. Native SpaA was similarly prepared from the supernatant of cultures of Strep. sobrinus strain FRID. Recombinant S A I / I I (r-SAI/II) was prepared from the cellular fraction of cultures of

Escherichia coli LC137 transformed with pSM2949 [7], and further purified by ion exchange chromatography and gel filtration as described for native preparations. The presence of S A I / I I or SpaA in final antigen preparations was confirmed by radioimmunoassay using monoclonal antibodies (mAbs) specific for S A I / I I and SpaA [15]. /3-Galactosidase (/3-Gal, G-6008, Sigma, U.K.) and bovine serum albumin (BSA, A-450B, Sigma) were used as control antigens. All antigen preparations were shown not to be mitogenic based on the lack of proliferation by normal PBMC and a control CD4 + cell line after 3 days of culture in the presence of antigens. 3.3. Interleukin 2 Lymphocult-T (TLF, 811020, Biotest AG, F.R.G.), a lectin-free supernatant produced by Phytohaemagglutinin (PHA) stimulated human T lymphocytes, was used as a source of interleukin 2 (stock solution 2 0 0 U / m l IL-2). 3.4. Cell separation Venous blood was obtained from healthy volunteers and defibrinated. Peripheral blood mononuclear cells (PBMC) were obtained by density sedimentation on Ficoll-Histopaque 1077 (1077-1, Sigma). The serum was removed and the cells washed twice in medium before being resuspended at 2 x 106 cells/ml in medium supplemented with 10% autologous serum. 3.5. Establishing cell line PBMC at 2 x 106/well were cultured in two wells of a 24-well plate (76-063-04, Flow Labs) in the presence of 10-20 ~ g / m l of S A I / I I (batchF166; 2 ml final volume). A control culture was set up in the absence of antigen. After 7 days cells were harvested and restimulated. Viable cells (0.5 x 106/well) were restimulated by 10-20 ~ g / m l S A I / I I together with 1-1.5 × 106/well of fresh autologous irradiated (40 Gy) PBMC (as a source of antigen presenting cells) in a 24-well plate (2 ml final volume). Restimulation with antigen and autologous PBMC was repeated every 7 days. T L F (5% final concentration) was included in the medium after two cycles of restimulation (added on days 1 and 5).

179

3.6. Assays for specificity and HIM restriction Specificity assays were performed at the end of a restimulation cycle. Line cells (104/well) were cultured with autologous irradiated P B M C (10S/well) in round-bottomed 96-well microtitre plates (1-63320, Nunc) together with antigen at a final concentration of 1 - 2 5 / ~ g / m l in a volume of 200 /~1. After 3 days, cultures were pulsed with [3H]thymidine (TRA120, Amersham International 9.25 kBq/culture) and 4 h later the wells were harvested onto glass fibre for liquid scintillation counting using a LKB fl-spectrometer. Restriction assays were performed in similar fashion but used irradiated PBMC from a panel of HLA-typed donors. 3.7. Blocking responses with anti-DR and DP rnAbs Specificity assays were performed in the presence of mAbs purified from supernatants containing anti-HLA D R and anti-HLA DP m A b s derived from the hybridomas L243 (HB55 obtained from ATCC, Rockville, MD), and B721 [23], respectively. These were used at dilutions of 1 / 4 1/400. An anti-class I m A b (HLA-A, B, C) purified from the supernatant of hybridoma W 6 / 3 2 (HB95 obtained from the ATCC, Rockville, M D ) was used as a control at dilutions of 0.2-20/Lg/ml. 3.8. Cytofluorimetric analysis T cells were phenotyped according to a method previously described [24] by staining with murine monoclonal antibodies to leucocyte c o m m o n antigen (LCA, CD45), CD3, (OKT3), CD4 (OKT4), CD8 (OKT8) and the c~fl T cell receptor (7770, Becton Dickinson, Mountain View, CA, U.S.A.) followed by an FITC-conjugated goat anti-mouse I g G (9031, Becton Dickinson). The presence of the ~,6 T cell receptor was investigated by means of a direct F I T C conjugate (TA2061, T cell Sciences Inc., Cambridge, MA, U.S.A.). Cytofluometric analysis was performed using a FACS scan (Becton Dickinson).

Antigen (ug/ml) Med SAI/II 20 10 5 2.5 1.3 B Gal

m

B !

20 i 5

i 10

i 15 [:)prn (x1000)

i 20

i 25

30

Fig. 1. Proliferative response of the SAI/II reactive T cell line to varying doses of native antigen (batch F166). B-Galactosidase (¢l-Gal) is given as a negative control antigen.

his dental plaque, and to have serum antibodies to S A I / I I , was used as a donor of PBMC. A cell line was established by culture with native, purified S A I / I I as described above. After three cycles of restimulation with antigen, there was a 3-4-fold increase in cell number with each cycle. The line was tested for specificity and shown to respond in a dose-dependent manner to S A I / I I and not to a control antigen, B-Gal (Fig. 1).

4.2. Antigen specificity The specificity of the line was not confined to the original batch of antigen used in its isolation, as it proliferated against two other batches of S A I / I I (F167 and F186) prepared by the same A n t i g e n (ug/rnl)

Med

SAI/II 20

r-SAI/II 20

10

t I

5

2.5

4. RESULTS

4.1. Isolation of a S A I / H specific cell line A normal healthy male volunteer shown to contain both Strep. rnutans and Strep. sobrinus in

i 4+

I 6

f I 8 10 Dpm (xl000)

i 12

i 14

i 16

Fig. 2. Proliferative response of the SA1/II reactive T cell line to varying doses of a recombinant antigen. The response to native SAI/II is given for comparison.

] 80

Table 1 Proliferative response to SAI/I1 and to SpaA antigens Antigen/batch Medium j3Gal ~ SAI/II/F166 /F167 SpaA/F132 /F140

Conc. (#g/ml)

Expt. 1 Dpm _+SD ~ (SI) b

Expt. 2 Dpm -+ SD (SI)

Expt. 3 Dpm -+ SD (SI)

25 d 10 e 10 10 10 e

1959_+ 100 (1.0) 1959_+ 100(1.0) 17 768 _ 1308 (9.0) 21 841 _+ 928 (11.1) 2574-+ 229 (1.3) 2826_+ 470 (1.4)

1264_+ 68 (1.0) 1796-+ 77(1.4) 12 658 _+803 (10.0) 12997-+244 (10.3) 1871 _+547 (1.5) 4242_+244 (3.4)

1757+ 478 (1.0) 2122-+ 92(1.2) 21000_+ 1 694 (11.9) N.D. r N.D. 4229_+ 642 (2.4)

Mean dpm of 3 replicate cultures+_ 1 S.D. b Stimulation index = Mean experimental d p m / M e a n medium dpm. /3-galactosidase. a Except for Expt. 1 (7/~g/ml). Except for Expt. 3 (20 /~g/ml). f Not done.

procedure from the supernatants of cultures of Strep. mutans. Similarly, S A I / I I obtained by extraction of the bacterial cell pellet with urea also elicited specific proliferation in three separate experiments (results not shown, e.g, 17050__+ 815 dpm compared with a background of 2897 +_ 134). Further evidence that the response was specific for S A I / I I was shown using a recombinant antigen (r-SAI/II). Because r-SAI/II is not readily released into the supernatant, it was extracted and purified from the pellets of E. coli LC137 expressing the spaP gene [7]. The results shown in Fig. 2 demonstrate that r-SAI/II and native S A I / I I produced comparable levels of stimulation. Specificity of the response was also investigated using SpaA, the Strep. sobrinus homologue of SAI/II. Two different preparations of SpaA (F132 and F140) generated little or no response in comparison to equivalent doses of the Strep. rnutans antigen (Table 1).

cules were performed. Fig. 3 shows that antigen specific responses were inhibited by anti-DP and anti-DR mAbs. In contrast, antibodies to HLA class I molecules had a minimal effect on proliferation. To attempt to map the HLA restriction directly, a panel of donors typed for HLA-A, -B, -C and DR specificities were employed. Data for the S A I / I I reactive line and a control line, previously Blocking antibody F+L+SAIBI

1/400

Anti-DP

~

29

1/40 1/400

Anti class I (ug/rnl)

4. 3. Phenotype and analysis of HLA restriction The SAI/II-responsive line was phenotyped by FACS scan using a panel of mAbs to T lymphocyte surface antigens and shown to be exclusively CD3 +, CD4 +, C D 8 - and TcR aB + yS-. This phenotype strongly suggests that the response would be restricted by class II MHC molecules. To investigate this further, inhibition studies with mAbs directed against HLA DR and DP mole-

1/40

Anti-DR

20 2

~

34

I1' 0 20

40 60 % specific inhibition

MII

Ilg)

Fig. 3. Inhibition of proliferation by monoclonal antibodies to class I and class II HLA molecules. Two dilutions of anti-DR, anti-DP or anti-class I monoclonal antibodies were used. Positive control responses were obtained using line cells (L) and autologous antigen presenting cells (F) together with S A I / I I antigen. Percent specific inhibition (values shown) was calculated from the formula. 1 - ((experimental d p m - b a c k g r o u n d dpm)/(maximum positive control d p m - b a c k g r o u n d dpm)) x 100.

181 Table 2 HLA-DR restriction of the proliferative response to SAI/II APC a

Autologous FF AC DJ DD

DR haplotype b

DR3 DRw52 a,b DR3 DRw52 a,b DR5, DR3, DRw52b DRV, DR3, DRw52b DR7

Responseof T cell line c SAI/II a

Autoreactive e

8.3_+3.2 2.9+_0.4 1.5 1.5 +0.2 1.4+_0.1

14.8 18.5 10.2 15.2 2.7

a Sourceof antigen presenting cells. b The HLA DRw52 variant (a or b) was determined for FF, AC and autologous APC by polymerase chain reaction amplification followed by restriction fragment length polymorphism and labelling with a specific probe. c Proliferative response given as stimulation indices+ 1 S.D. a Response of SAI/II specific line in the presence of SAI/II and antigen presenting cells. Mean of three separate experiments except AC (tested once). e Response of autoreactive line to antigen presenting cells. Results from one experiment. found to respond directly to autologous PBMC in the absence of extraneous antigen, are shown in Table 2. Whereas the autologous response mapped to HLA-DR3, the results obtained with the antigen-specific line were not so clear. Small but reproducible stimulation was always observed using cells matched from the DR3 supertypic specificity DRw52a but not DRw52b.

5. D I S C U S S I O N Streptoccocal antigen I / I I ( S A I / I I ) has been extensively investigated and shown to have a number of important functions including the sucroseindependent attachment of Strep. mutans to the tooth surface [11]. Systemic immunisation with S A I / I I in an animal model induces specific serum antibodies [25] which can be expressed at the tooth surface in gingival crevicular fluid [26]. A reduction in the level of Strep. mutans colonisation as well as lower caries scores have been demonstrated in rhesus monkeys systemically immunised with S A I / I I [25]. Furthermore, murine mAbs raised against S A I / I I have been successfully used in local passive immunisation of both rhesus monkeys [28] and human volunteers [29].

mAbs raised against the SpaA protein of Strep. sobrinus which cross-reacts with S A I / I I from Strep. mutans, have also been used successfully in human studies [30]. T lymphocyte responses have also been studied extensively using PBMC from actively immunised monkeys and naturally sensitised human subjects [20,25]. These studies have involved the characterisation of helper and suppressor functions elicited by S A I / I I [31[, as well as proliferative responses to a synthetic peptide corresponding to the sequence deduced from a cleavage fragment [20-22]. Identification of major T cell epitopes is greatly facilitated by the isolation of antigen-specific T lymphocyte lines or clones. The CD4 + S A I / I I specific line reported here was derived from a naturally sensitised normal individual using standard procedures; a similar strategy has been employed to isolate CD4 + clones and lines specific for fl-Gal [32]. The frequency of T cells responding to SA1/II, in the peripheral blood, has been estimated using limiting dilution analysis and found to range from about 1 : 3 8 0 0 0 to 1 : 8 6 0 0 0 [33]. This lower frequency limit was, in fact, the frequency of SAI/II-reactive T cells in the blood of the donor used to establish the line (95% confidence limits 1 : 67 000-1 : 110 000). If it is assumed that T cells represent 70% of the blood leukocytes then the line may have originated from as few as 25-42 cells. Specificity assays showed that the line responded to both S A I / I I and a recombinant S A I / I I protein, but not to the SpaA protein from Strep. sobrinus. Nucleotide sequencing of pSM2949 indicated that the recombinant plasmid did not contain the entire spaP gene [16]. This results in the expression of a truncated polypeptide of rS A I / I I of M r 155 000 comprising residues 1-1300 of the native polypeptide. The recombinant antigen originated from a different strain of Strep. mutans (NG5, as opposed to Guy's) and indicates that the epitopes recognised by the line are conserved between at least two strains. Moreover these T cell epitopes are not within the 253 amino acid residues at the carboxy terminus as these are absent from the recombinant product [16]. Furthermore, Takahashi et al. [17] have shown significant homology between PAg from Strep. sobrinus

182

and the analogous antigen (PAc, SAI/II) from Strep. mutans to be restricted to residues 612-919 of the Strep. mutans molecule, this suggests that the specificity of the line could lie outside this region. Further experiments will be required to verify whether a lack of response to SpaA is a general feature and how differences between individuals, notably their HLA haplotype, influence the response. The inhibition studies using mAbs indicated the presence of cells restricted by HLADR, probably DRw52a, and HLA-DP within the line. A minority of T cells restricted by HLA-DQ might also be present. To precisely determine the epitope specificity of the line we hope to generate smaller fragments of the r-SAI/II and corresponding overlapping peptides, an approach which should permit the identification of cross-reactive T cell epitopes which will be of importance for the design of effective vaccines against dental caries.

ACKNOWLEDGEMENTS We wish to thank J. Cridland and T. Beck for the careful preparation of the manuscript and Dr. C. Kelly for letting us use the pSM2949 construct which originated from Dr. A. Bleiweis. Furthermore, the authors wish to thank R. Vaughan for doing the tissue typing and to J. Lanchbury and P. Walker for helpful suggestions and discussions.

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