rmmunol. Cell Biol. (1990) 68. 299-305

Identification of antigens which stimulate T lymphocytes of Salmonella enteritidis llRX immunized mice Hans-Martin Vordermeier* and leva Kotlarski Department of Microbiology and Immunology, The University of Adelaide, Adelaide, SA 5000, Australia (Submitted

10 May 1990. Accepted for publication 4 October 1990.)

Summary The technique of using sodium dodecylsulfate-polyacrylamide gel electrophoresis fractionated antigens (Ag) transferred to nilroccllulose filters was adopted to analyse T eell responses to Salmonella cnteritidis 11RX Ag. Employing in vitro proliferation assays wilh T cells from S. cnterilidis 1 lRX-primed (BALB/c XC57BL/6J)F I mice as the measure of T cell stimulation, we have identified Ag able to stimulate T eells in the regions eontaining 16,24.34 and 50-60 kDa proteins, wilh dominant Ag aelivjty al about l6kDa. These results were confirmed with long-term. Ag-speeific L3T4+ T cell lines which responded lo molecules in ihe same four Mr regions, suggesting that no selection by a single aniigenic determinant had occurred during more than 3 months of in viiro culture, or that all the molecules which were stimulatory shared al least one anligenie determinant. Beeause the seven clones we examined responded only to 16 kDa molecules, the former alternative is the more likely. Standard immunoblol analysis indicated that these Ag also act as major B cell stimulating determinants. T cells of BALB/c mice, which are 5-10 times more resistant to 5. enteritidis I lRX than C57BL/6J mice, showed the same pattern of reaetivity as Fl mice whereas the major antigenie region for T cells of C57BL/6J miee was located between 50 and 60 kDa.

INTRODUCIION Previous studies in our laboratory have shown that immunization with the 1IRX strain of Salmonella enteritidis (I IRX) provides resistance to mice against lethal infection with the highly virulent Salmonella typhimiiriuni C5. The response is T cell-mediated and T eells from 1 IRX-immunized animals can be induced to proliferate//) r/Vwaswell as release lymphokines when cultured in the presence of antigen (Ag)presenting cells and formalin killed 11 RX (fllRX) organisms or soluble I IRX (sllRX)

Correspondence: I. Kotlarski. Department of Microbiology and Immunology, The University of Adelaide, SA 5000. Australia. 'Present address: MRC Tuberculosis and Related Diseases Infeetions Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, UK. .Abbreviations u.ved in f/jf'.v paper: .\g, antigen: DTH,

delayed type hypersensilivity: FCS, felal ealf scrum: Fl miee, (BALB/c X C57BL/6J)F1 mice; fl IRX. formalin killed llRX: i.p.. intraperitoneally: IPC. PC from immunized mice; MoAb, monoclonal antibodies: MHC. major histoeompatibilily complex: NPC, PC from normal mice: PC. peritoneal cells; 11 RX, Salmonella emeritidis i 1 RX.

antigens (1-3). The responding T cells are mainly L3T4+ T cells and are likely to represent a mixed pool with a range of different antigenie specificities, reflecting the Ag preparations used to stimulate their responses. The present study was designed to define the Ag of 1 IRX which can induce proliferation of these immune T eells. As part of this approach, we adopted a recently described technique that uses the resolving power of sodium dodecyisulfate-polyacrylamide gel electrophoresis (SDSPAGE) to fractionate Ag into discrete bands and allows the probing of the specificity ofprimed T cells by measuring Ag-speeific T cell responses to the separated ant igens after electroblotting them onto nitrocellulose (4). We have identified Ag recognized by T cells in suspensions freshly prepared from animals immunized with 1 IRX and by well-established L3T4 * T cell lines and T cell clones prepared from these T cell suspensions by long-term culture with fllRX. MATKREALS AND METHODS Miee BALB/c. C57BL/6J, and (BALB/c X C57BL/6J)FI (Fl) mice were obtained from the Central Animal House, The University of Adelaide,

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Culture media Hank's balanced salt solution supplemented with 100 IU of penicillin/mL and 100 |ig of streptomycin/mL was used to harvest and prepare cell suspensions. RPMI 1640 (Difco) with 2 mmol/L glutamine. O-I mmol/L 2-mercaptoethanol. 5 |jg of indomethacin/mLand 10%(v/v) heat-inactivated fetal calf serum (FCS) (Flow Laboratories. Sydney. Australia) and antibiotics was used for all cell culture work and will be referred to as culture medium. Bacteria and baeterial Ag Overnight cultures of S, cnteritidis llRX (llRX) were diluted 1/10 in nutrient broth and grown for 3 h with shaking at 37°C to give approximately 2X 10'' live bactcria/mL, For routine immunization of FI mice, the cultures were then diluted in cold saline and 10^ bacteria per mouse were injected intraperitoneally (i.p.). n i R X were prepared by treating the baeteria at iC/mL with 1% (v/v) formalin for 1 h at 37^, The bacterial suspension was adjusted to I mg/mL with saline after determination of its dry weight and suitable aliquots were stored at — 2Q°C. The final concentration of fl 1RX used for in vitro proliferation assays was always i |ig/mL. Lymphoid cell suspensions. T cell lines and clones Peritoneal cell (PC) suspensions from Fl mice Immunized wiih 1 I RX 14 days earlier (IPC) provided the source of primed T cells. Before use, the IPC was passaged over nylon-wool columns and ireatcd with anti.-Ia'' monoclonal antibodies (MoAb; ascites fluid from HB3 hybridoma) and complement {I h al 3TC at a density of 10' cells/mL) as previously described (3). Peritoneal cells from normal mice (NPC) were used as Ag-presenting cells. T cell lines were established from IPC harvested from mice immunized with 11 RX. They were maintained by alternating cycles of stimulation with fl lRX antigen (1 |jg/mL) and niitomycin C-trcaled spleen cells and rest without antigen. Clones were established by limiting dilution cloning from lines after at least ihe ihird cycles of stimulation and rest (5). in \'HTO proliferation assays These were set up in quadruplicate in flat-bottom 96 well trays (Linbro 76 033 05) at a cell density of 10^ IPC and 2X10" NPC or 2- 5 X 10^ T cells from lines or clones with 2X 10" NPC in 0-2 mL portions in culture medium. Cultures were pulsed with ['H]-thymidine. 37 kBq per well: Amersham) after 3 days (unless indicated otherwise) and harvested and processed after 4 h of additional incubation, as described earlier (3), Fluorescence-aetivated eell sorter (FACS) analysis The phenotype of the T cell lines and clones was established by FACS analysis using MoAb specific for the T eell markers Thy 1 -2 (TIB 107). L3T4 (TIB 207) and Lyt 2-2 (TIB 150), Detailed information about these MoAb and the method used to label the cells have been provided elsewhere (.'^.6), The FACS machine used was a Becton Dickinson FACScan,

Local transfer of lymphoid cells and measurement of footpad swelling Cells from cultures of T cell lines and clones were suspended to the required concentrations in culture medium without FCS and antibiotics. T cell lines (5X l05celis)orTcellctones(2X 10^ cells) inO'O5 mL were injected alone, or with fl IRX (2-5 jjg) or live IIRX (10^) into the right fiind footpads of recipient miee and the thickness of both hind feet of the injecled mice were measured at various times ihercafter, using dial gauge calipers with accuracy to 0' i mm (5.7). Preparation of ami-] IRX serum Mice were immunized by i.p. injection of 10-^ live 1 IRX organisms, followed by three weekly i.p. injections of 0-5 mgof fl IRX. starting 4 weeks later. They were bled 1 week after the last injection and serum was prepared and stored in small aliquots in the usual way. SDS-PAGE and immunobhtting Fl 1 RX antigens were analysed by SDS-PAOE using 14 and 15'Ki polyacrylamide gels in the system of Laemmli (8), Bands were stained with Coomassie Brilliant Blue, using standard procedures. Immunostaining was performed after transferring protein bands onto nitrocellulose filters (9), A mouse anti-llRX serum (1 : 500) and goat immunoglobulins specific for all mouse immunogtobulin classes and coupled to alkaline-phosphatase (Sigma, I ; 30(1) were the staining reagents used. Preparation of fractionated niiroeellulose-bound Ag For the characterization of I IRX Ag 50 ^ig of fl 1 RX were loaded per well of a 15% SDS-PAGE gel which was run under reducing conditions and electroblotted onto nitrocellulose filters. The filters were cut into sections, solubilized with dimethylsulfoxide and, after 1 h ineubation at room temperature, were precipitated by addition of an equal volume of 0^05 mol/L carbonate/bicarbonate buifer pH 9-6, Tbe precipitates were centrifuged. washed three times with RPMI 1640 and each suspended in 1 mLofculture medium before storing at —2O''C; 25-40 ^L volumes of these suspensions were used in in vitro proliferation assays (4,10).

RESULTS Response ofprimed T cells to fractionated I IRX antigens To define the Ag of S. enteritidis 1 IRX which are able to induce proliferation of 1 lRX-primed T cells, we prepared suspensions of nitrocelltilose-bound Ag which had been fractioned by SDS-PAGE before being eleclroblottcd otito nitrocellulose fitters for use in T cell proliferation assays. Figure 1A shows a SDS-PAGE profile of the separated fl IRX Ag preparation after staining with Coomassie Blue. Results of proliferation assays using purified T cells of mice

IDRNTIFICATION O¥ SALMOMILL.A ANTIGRNS

301

100

80

-

60

•40

20

66

4b

24

14,2

Antigen size (kDa)

S A

B

Fig. 1. SDS-PAGF analysis of f 11 RX antigens. (S) Standard proteins: (A) SDS-PAGE (14%) profile of separated f l I RX antigens afler staining witfi Coomassie Blue: (B) immunoblot of the same preparation, stained using mouse anti-1 IRX serum.

immunized with I I R X indicated a dominant antigenic region around 16 kDa and other antigenic determinants at 25. 34 and between 50 and 60 kDa (Fig. 2). In contrast, a standard immunoblot stained with mouse a n t i - l l R X serutii (Fig. IB) indicated that imtiiunization with I I R X induced the production of antibodies against virtually every protein in the preparation, with major bands being located in the molecular range above .'^5 kDa and below 20 kDa. Thus, it appears that the inajor T eell stimulating Ag located in the 16 kDa region act as tnajor B cell stimulating determinant(s) as well. This could prove very useful for the purification and detailed eharacterization of these Ag.

Response of T eell lines ami elonvs of T cells lo fraetionated fl IRX antif^'ens To determine whether long-term culture of T cells preferentially selected T cells whieh react

Fig. 2. Tcell stimulating ability ofSDS-PAGF fractionated fl IRX antigens, assessed by measuring ['H]thymidine uptake hy 1 IRX-primed T cells cultured for 4 days with NPC and nitrocellulose-bound antigens and pulsed with ['HJ-thymidine tor 4 h before harvest. Cells cultured in medium only incorporated 2 5 7 3 ± I 3 7 ct/min: f M R X (I ng/mL) induced a response of 121 733 ± 3667 ct/min.

with major antigenic determinants of 1 IRX. a comparison was made between the 'stimulation profile' obtained when fractionated, nitrocellulose-bound f l IRX antigens were cultured with primed T cells freshly isolated from I I R X immunized mice and the profile obtained using three long-term 1 IRX-specifie T eell lines and seven T eell clones. T cell lines were established by eulturing 1 IRX-primed, purified T cells of Fl tnice in alternating cycles of stimulation with Agpresenting cells and fl IRX tbilowed by culture without Ag. All three lines proliferated specifically in response to f l IRX antigens and were characterized as L 3 T 4 \ Thy 1 ' . Lyt 2~ cells (Table la). They could also be used to transfer delayed type hypersensitivity (DTH) against fortiialin-killed as well as live 1 IRX organistiis to unpritiied recipient mice (Fig. 3A). When these T ceil lines were cultured with the nitrocellulose-bound fractionated f l l R X anligens. a pattern ot" ptolileration similar to that observed using freshly purified primed T cells was obtained. Because the tesponses ol tlie three T cell lines wore virtually identical, the tesults tot only one of them (line P2) are shown in Fig 4.

H-M. VORDERMEIER AND 1. KOTLARSKI

302

Table 1. Characterization of 5, enteritidis I IRX spccitic T cell lines and T cell clones. [3H]-Thymidine incorporation (ct/niin)"^ aflcr 3 days of culture of:* T cells tested*

T cells+ f IIRX

T cells + NPC

Tcclls + NPC + fllRX

(A) T cell lines G2 P2 O3

397 + 345 3191 ± 823 153± 46

3079 ± 3079 13 602± 477 5246 ±261 5

627t6± 3117 121 602 ±29 104 87 940 ± 9879

NPC + fllRX I 990 ± 425 642± 31 642± 31

(B) T cell clones 196± 43 24 291 ± 2248 41B3 87 ± 65 t559± 137 993 ± 377 67 466 ± 2319 32A3 207 + 51 I587± 676 476± 72 106 322 ± 1522 51C2 1063 ± 231 5376± 1344 476+ 72 108 894± II 617 52B1 70 ± 3 1493+ 295 *T cell lines and clones cullured with medium alone always incorporated ct/min < T cells + f 1 I RX, ^Mean ct/min of four dcterminalions ± s.d. tT cells and NPC(3X lO'* per well) alone or mixed together were cultured in 0-2 mL portions, with or without fllRXd TO-t

10

24 h

46 t)

Fig. 3. Local transfer of DTH to 11 RX anligens. (A) Using an Ag-specific T cell line; (B) using an antigen-specific T cell clone. Cells 5 X 10^ from line P2 or2 X 10^ cells from clone 52B1 were injected into the right hind footpad of three normal mice alone or with f 11 RX or live 11 RX (11X) organisms. Control groups, injecled with Ag alone were also included. The per cent footpad swelling data shown are Ihe calculated mean values, from measurements of both hind feet of three mice per group. Stimulatory molecules were detected with Mr of approximately 16. 25, 35 and between 50 and 60 kDa (cf. Fig. 2). Seven T cell clones were derived from longterm. 1 lRX-specific, L3T4+ T cell lines by limiting dilution cloning. They all proliferated when cultured with f l l R X (I |ig/mL) and the response of four of these clones is shown in Table lb. The results illustrate the range of variation in proliferation seen with the ditferent clones: repeated testing of individual clones established that the response of each clone was

quite stable and characteristic of the clone. The clones were also able to transfer a DTH response to unimmunized recipients and the results obtained with clone 52B1 are shown in Fig. 3B. When cultured with the nitrocellulose-bound fractionated fl IRX antigens, all the T eell elones responded only to molecules in the 16 kDa region. The proliferation profiles obtained with clones 32A3and 41B3 are shown in Fig. 5. They are typical of the proliferation histograms obtained with the other T cell clones tested. Pre-

IDENTIFICATION OF SALMONELLA ANTIGENS

303

50

.-.

40-

.2

10

n

14.Z

Anllgen size (kDa)

FiR. 4. The ability of SDS-PAGE fractionated, fl lRX antigens to stimulate line P2 T cells, assessed by measuring ['H]-thymidine uptake as described in Fig. 2.

Antigen size (hDa)

Fig. 5. The ability of SDS-PAGE fractionated. rilRX antigens lo stimuiale T coll clones 32B3 (hatched) and 41 B3 (unhalchcd), assessed hy measuring t-'H]-thymidinL' uptake as described in Fig. 2.

sumably. these findings emphasize the immunogenicity of IIRX antigen{s) in the 16 kDa region. Characlerization of the spccificiiy of 1IRXprimed T cells of different .strains of mice In all the studies deseribed above we used Fl mice as a source of primed T cells. Their parent strains {BALB/c and C57BL/6J) show a diflercnce in susceptibility towards IIRX. with the LDso for BALB/e mice {H-2'') being 5-10 times higher than the LD5() for C57BL/6J mice {H-2'') and about the same as for Fl mice (7). This raises the possibility that the two parental strains may not respond to the same antigetiic determinants, a possibility supported by our earlier observation that when Fl mice are immunized with live 11RX. a 'preferred* association of IIRX antigens with iI-2'' haplotypc products can be demonstrated (3.7). Aecordingly. we eompared the 'stimulation profiles" obtained when purified. 1 IRX-primedT eells of C57BL/6J. BALB/e and Fl miee were cultured with nitrocellulose-bound fractionated fllRX Ag. In contrast to Fl and BALB/c mice, where the 16 kDa Ag are immunodominant. the major antigenie region in C57BL/6J miee was located between 50 and 60 kDa (Fig. 6). However, the overall prolilerative response ofC57BL/6T cells was lower than that measured for Fl and BALB/e T cells (data not shown).

46 - F l IPC

24 - • - C57BI6J tPC

Antigen Size (kDa)

hig. 6. The abiliEy of SDS-PAGE fractionated. fl IRX antigens to stimulate 1 IRX-primed T cells of Fl and C57BL/6J mice, assessed by measuring ['Hjthymidine uptake as described in I-ig. 2.

DISCUSSION The technique of using SDS-PAGE fraetionated Ag transferred to nitrocellulose filters was adopted to analyse T cell responses to 11 RX Ag. The advantages ol" this technique are that biochemical purification of individual antigens is

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H-M, VORDERMEIER AND I. KOTLARSKI

not required, the antigens do not have to be preselected (e.g. by serological techniques) and it has been proven to be successful for the detection of various mycobaeteria! antigens (11-13). Using in vilro proliferation assays with T cells from 1 lRX-primed mice as the measure of T cell activation, we have identified antigens able to stimulate Teells in regions containing 16. 24. 34 and 50-60 kDa molecules. We confirmed these results using long-term L3T4"' T cell lines specific for S- cnlcriiidis I IRX. The lines responded to molecules in the same four M; regions, indicating either that no seleetion by a single antigenic determinant had occurred during more than 3 months of//I r/7m culture or that all the molecules which were stimulatory shared at least one antigenic determinant. Because the seven clones we examined responded only to 16 kDa molecules, the former alternative is the more likely. This is supported by our finding that absorption of mouse anti-11 RX serum with SDS-PAGE fractionated antigens of Mr greater than 16 kDa did not remove antibody activity for the 16 kDa antigen(s) (data not shown). Although not much is known about the nature of Salmonella antigens which stimulate T cells, it is possible to compare our findings with the results of others who have analysed various bacterial extracts (14.15) and isolated bacterial proteins to establish their protection-inducing properties. Isibasi ct al. (14) were able to protect mice against challenge with Salmonella lyphi by immunization with an outer membrane protein preparation of the same organism. It is interesting to note that their preparation consisted of a range of proteins with Mr 17. 23-28 and 34-41 kDa. corresponding quite closely to the antigens we have defined in our system — molecules of Mr 50-60 kDa are the only ones not included in their preparation. A difference between their results and ours is that we detected a strong humoral response to molecules in the 16 kDa region, whereas they reported significant humoral responses only against antigens in the 36 and 4! kDa region. Because we used whole bacteria instead of bacterial extracts to induce a humoral response, It is possible that the low molecular weight antigens may induce humoral responses only when injected as an integral part of whole bacteria or with an adjuvant. The proteins in the region around 34 kDa eould be porins or the heat-modifiable protein OmpA. We favour the former beeause they have been shown to be effeetive in eliciting a cellular immune response in murine salmonellosis (16). There is also a report describing the protective efieet of a 55 kDa protein from Salmonella

typhimwium, also characterized by immune serum, which may correspond to a stimulating protein we have defined in the 50-60 kDa range (17). However, the antigenic activity we detected in the 50-60 kDa range could also be due to heat-shock protein which is widely found in bacteria (18). Consistent with this possibility is the report of Gaston and coworkers (19) that T cells from arthritis patients which responded to purified protein derivative and the 65 kDa heatshock protein from Mycobaeterium leprae were also stimulated by a 65 kDa protein from Salmonella agoni. Murine and human L3T4^ T cell clones specific for various bacterial antigens of a number of different organisms including Lisferia. Mycohacierlae. Shigella Jle.xneri and Yer.sinia enterocolitica (5.20-22) have proved to be invaluable tools in studying the immunobiology of these organisms. The T cell lines and clones described in this study are likely to be just as useful. They have already been helpful for the identification of the 11 RX antigens whieh are able to induce //; vitro proliferation of T cells with demonstrable biological function in vivo, and therefore should assist the identification of antigens with protective potential. Derivation o^ additional 1 IRX antigen-specific T cell clones should permit a clonal analysis of the T cell response to S. enterilicli.s, as has been done for the B-cell repertoire (23). Despite the fact that all of the seven T eell clones we have analysed so far respond only to the !6 kDa antigen(s). we anticipate that future cloning experiments will provide clones specific for all of the other I IRX antigens we have identified. It should then be possible to assess the relative importance of the various antigens in protection and to determine their biological significance in vivo. The difference in response to 1 IRX antigens by T cells of BALB/c and C57BL/6J mice may indicate the existence of a hierarchy of "protective' antigens. Although we realize that other explanations are available, it is tempting to suggest that C57BL/6J mice are more susceptible to I IRX infection than BALB/c (and Fl) miee because they fail to mount a major response to the 16 kDa antigen(s) due to poor association of these molecules with major histocompatihility complex (MHC) coded products of the /7-2* haplotype. However, since the genetic control of susceptibility/resistance to various intracellular bacterial parasites has been mapped to the hy locus (24). more data are required before any firm conclusions can be made. With this in mind, we intend to analyse the ability of T cells of other inbred strains ot" mice (with different

IDENTIFICATION OF SArXfONLLLA ANTIGENS MHC haplotypes) to respond to nitrocellulosebound I I R X antigens prepared from I I I R X . Our interest in using this approach has been raised by the recent report ot considerable differences in response to nitroecllulose-tVaetionated M. leprae antigens by T eells of family contacts and patients with leprosy (13),

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11. Lee. S. P..Stokes. W. G..Grant.K.A.(Va/. 1989. Cellular immune responses of leprosy contacts to fractionated Mvcohaclerium leprae antigens. Infea. Immun. 57: 2475-2480. 12. Mehra. V.. Bloom. B. R.. Torigan. V. K. et al. 1989. Cliaracteri7ation of Mycohacterium leprae cell wall-associated proteins with the use of T lymphocyte clones. ./. Iinmtinol. 142: 28732878. .4fkii(nrledf;emi'nf.s We thank Mr G. Penney for 13. Mendez-Sampiero. P.. Lamb. J.. Bothamby, G. excellent technical assistance. H-M.V. was the reelal. 1989. Molecular study of the T cell repertoire cipient ol'a University of Adelaide Medical Research in family contacts ynd paiienls with leprosy. J. .•\ssociateship and this work was supported by a Immunol. 142: 3599-.1604. University of Adelaide and a NH&MRC Research 14. Isihasi. A..Ortiz, V.. Vargas. M. ('/(//. 1988. ProGrant. tection against Salmonella lyplit infection in mice after immunization wiih outer membrane proteins isolated from Salmonella rvplii 9. 12. d, Vi. Infect. Immun. 56: 2953-2959. 1. Atiridgc. S. R. and Kotlarski, I. 1985. In viirv 15. Udhayakuniar. V, and Muthukkaruppan. V. R. lymphokine release hy lymphocytes from mice 1987. Protective immunity induced by outer infeeted with Salmonella. .Aiist.,/. E.xp. Biol. Med. membrane proteins of Salmonella typhimunum Sri. 63: 4X9-502. in mice. Inject. Immun. 55: 816-821. 2. Davies. R. and Kotlarski, 1. 1976. The role of 16. Udhayakuniar, V. and Muthukkaruppan. V. R. thymus-derived eells in immunity to Salmonella 1987. An outer membrane protein (porin) as an infection, .-iti.sl. ./. E.\p. Biol. Med .Sd. 54: 207eliciting antigen for delayeti-type hypersensitivity 219. in murine salmonellosis. Infect. Immun. 55: 8223. Kollarski. I., Pope. M.. Doherty, K. and Altridge. 824. S. R. 1989. The in vitro proliferativc response of 17. Foulaki. K.. Gruber, W. and Schlechl, S. 1989, lymphoid cells of mice infected with Salmonella Isolation and immunological characterization of a enleriiiih.s ! tRX. Immunol. Cell Biol. 67: 19-29. 55 kDa surface protein from .Salmonella typhi4. Ahou-Zeid. C. Filley. H.. Stecle, J. and Rook, mtirium. Infect. Immun. 57: 1399-1404. G. A. W. 1987. A simple new method for using 18. Young, D. B.. Ivanyi. J.. Cox. J. H. and Lamb, antigens separated hy polyacrylamide gel electroJ. R. l987.The65kDaantigenofniycobacteria — phoresis to stimulate lymphocytes in vilw after a common bacterial protein? Immunol. Today S: converting bands cut from western blots into 215-219. antigen-hearing particles. / Immunol. Method.y 19. Gaston. J. S. H.. Life, P. F.. Bailey, L. C. and 98: 5-10. Bacon. P. .A. 1989. In vitro responses to a 65 kDa 5. Boom. W. H.. Husson. R. N.. Young. R. A., mycobaclerial protein by synovia! T eells from David. J. R. and Picssens. W. F. 1987./H V7IY» and inflammatory arthritis patients, y. Immunol. 143: tn vitro eharaeterisalion of murine T eell clones 2494-2500. reactive to Mvcohactertum luberculosi.s. Infeci. 20. Kaufmann, S. H. E. and Hahn, H. 1982. Biological tmniun 55: 2223-2229. functions of T cell lines with specificity for the 6. Ashley. M. P. and Kotlarski. I. 1987. M Wiw H-2K intraeellular bacterium Listeria monocytof-cnes and H-2D antigen expression in two allogeneic in vitro and m vivo. J_ E.\p. Med. 155: 1754mouse tumours of low immunogenicity. Au.'H. J. 1765. E\p. Biol. Med Sci. 65: 323-.128. 21. Hermann, E., Fleiseher, B., Mayet, W. J. el al. 7. Attridge.S.R. and Kotlarski. I. 1985. Localtrans1989. Response of synovial fluid T eell clones to fer ofdelayed-typc hypersensitivity after .S'u/monYersinia enterocolilica antigens in patients with (•//(/ infection in miee. Infect. Imniiin. 50: 807reactive Yersinia arthritis. Clin. E.xp. Immunol. 812. 75: 365-370. 8. Laemmli, U. K. 1970. Cleavage structural pro22. Zwillich. S. H.. Duby. A. D. and Lipsky, P. E. teins during the assembly of the head of bacterio1989. T lymphocyte clones responsive to Shigelta phage T4. Nature 227: 680-683. flexnen. J. Clin. Mierohioi 11: 417-421. 9. Ey. P. L. and Ashman. L. K. 1986. The use of 23. Metcalf. E.S.,Gaflrey.M. and Duran,L.W. 1987. aikaline-phosphatase-conjugated anti-immunoThe diversity of the secondary Salmonella typhiglobulin in polyacrylamide gels. Methods Enzvm»;/Hm-specific B cell repertoire. ./. Immunol. inol 121: 497-509. 138: 3461-3467. 10. Lamb. J. R. O'Hehir. R. W. and Young. D. B. 24. Plant,J.andG!ynn..A. A. 1987. Geneliesof resist1988. The use of nitro-eellulose immunoblois for anee to infection with Salmonella lyphimurium in the analysis of antigen recognition by T mice. / Infect. Di.s. 133: 72-78. lymphocytes. J. Immunol. Methods 110: 1-10.

Identification of antigens which stimulate T lymphocytes of Salmonella enteritidis 11RX immunized mice.

The technique of using sodium dodecylsulfate-polyacrylamide gel electrophoresis fractionated antigens (Ag) transferred to nitrocellulose filters was a...
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