Immunology 1998 94 1–4

Vb11+ T-lymphocyte expansion by toxic shock syndrome toxin-1 differs in micebearing H-2q versus H-2b haplotypes

Y.-X. ZHAO,* U. BRUNSBERG,† R. HOLMDAHL† & A. TARKOWSKI* *Departments of Rheumatology and ClinicalImmunology, University of Goteborg, Sweden, and †Department of Medical Inflammation Research, University of Lund, Sweden

SUMMARY

We have recently demonstrated that toxic shock syndrome toxin-1 (TSST-1) expanded Vb11+T lymphocytes contribute to Staphylococcus aureus arthritis and sepsis-induced mortality.Interestingly, Vb11+ T-cell mediated joint pathology varies in different mouse strains. In thisstudy, we characterized the in vitro pattern of Vb11+ T-cell expansion by TSST-1 in mice withvarious genetic backgrounds. Mice expressing major histocompatibility complex (MHC) class III-E molecules did not expand Vb11+ T cells upon stimulation with TSST-1. Using B10 congeneicI-E negative mouse strains, we found that the TSST-1-expanded Vb11+ T cells in B10Q (H-2q)and B10M (H-2f ) mice but not in B10B (H-2b) mice. Antigen-presenting cells (APC) from B10Qmice, L cells and lymphoma cell line transfected with a q gene did not restore the deficient Vb11+T-cell expansion by TSST-1 in purified T cells from B10B mice. In contrast, I-Ab APC were ableto stimulate Vb11+ T cells from H-2q mice. Furthermore, Vb11+ T cells in H-2b mice did expandwhen exposed to staphylococcal enterotoxin A (SEA). These findings suggest that the T-cellrepertoire, skewed by clonal deletion and inactivation of self-reactive T cells, accounts for thedifferent magnitude of Vb11+ T-cell expansion among the different mouse strains.

INTRODUCTION antigens during T-cell ontogeny, have a profound effect onthe T-cell repertoire because of the clonal elimination of large

Superantigens (SAg) are a heterogeneous group of proteinsnumbers of potentially autoreactive T cells.2,4 There is athat induce T-lymphocyte stimulation in an extremely efficientsimilar usage of Vb families between T-cell response to away.1,2 The mechanism of cellular activation by superantigenscertain bacterial SAg and endogenous viral SAg. It has beeninvolves a selective and specific binding and presentation ofshown that Vb3 deletion is linked to mouse mammary tumourthese molecules on major histocompatibility complex (MHC)virus (Mtv) 1, 3, 6, 13, 44 infection, while partial Vb11 deletionclass II molecules to certain Vb elements on T-cell receptorsis associated with Mtv 8, 9, 11 in the presence of MHC class(TCR). Two classes of superantigens have been described,II I-E molecules.5 Thus, the Vb3+ and Vb11+ T cells fromnamely bacterial and endogenous. The bacterial superantigenssome mouse strains bearing I-E molecules do not respond toare toxins secreted by Gram-positive bacteria, such asstaphylococcal enterotoxin A (SEA), which is reactive withStaphylococcus aureus. The toxins are responsible for a varietyVb3+ and Vb11+ T cells.6of diseases in man and animal, including toxic shock and food

Toxic shock syndrome toxin-1 (TSST-1), a toxin secretedpoisoning.1 It has been shown that the pathogenic effects ofby Staphylococcus aureus, has been implicated in the patho-these toxins are mediated by hyperstimulation of T cells andphysiology of toxic shock syndrome.7 It has previously beencytokine production.3 The endogenous superantigens are enco-demonstrated that TSST-1 activates murine T cells expressingded by genes present in the genetic backgrounds of someTCR Vb3, Vb15, and Vb17 segments.1 Recently, we havestrains of mice. Endogenous superantigens, expressed as selfreported that TSST-1 contributed to Staphylococcus aureusarthritis8 by expansion of Vb11+ T lymphocytes in inflamed

Received 28 December 1997; accepted 31 January 1998. joints.9 Indeed, depletion of the Vb11+ T cells amelioratedAbbreviations: TSST-1, toxic shock syndrome toxin-1; SEA, arthritis and increased survival rate in Swiss mice.9

staphylococcal enterotoxin; Mtv, mouse mammary tumour virus. Subsequently, an in vitro study confirmed that TSST-1 prefe-rentially triggers Vb11+ T cells in Swiss mice, as characterized*Present address: The Toronto Hospital Arthritis Center, Theboth by their expansion and vivid interferon-c (IFN-c)mRNAToronto Hospital, 13-415 Mc, 399 Bathurst St., Toronto, Ontario,

Canada M5T 2S8. expression.10 Interestingly, Vb11+ T-cell-mediated jointpathology varies in different mouse strains,9,11 suggesting thatCorrespondence: Dr Y.-X. Zhao, The Toronto Hospital ArthritisVb11+ T cells in different mouse strains have different sensi-Center, The Toronto Hospital, 13-415 Mc, 399 Bathurst St., Toronto,

Ontario, Canada M5T 2S8. tivity in response to TSST-1. Thus, the difference of reactivity

© 1998 Blackwell Science Ltd 1

Y.-X. Zhao et al.2

of Vb11+ T cells to TSST-1 might be related to the T-cell Assay for Vb expression on T cells responding to superantigensThe splenocytes (2×106), the mixture of purified T cellsrepertoire skewed by clonal deletion of self-service T cells

caused by endogenous Mtv infection. To test the above (1×106) and mucosal mast cell (MMC )-treated spleen APC(1×106) or transfectants (5×104) were cultured in 24-wellhypothesis, the present study was undertaken to explore the

mechanism by which TSST-1-driven Vb11+ T-cell expansion culture plates (Nunclon, Roskilde, Denmark). The cells werestimulated with TSST-1 (10 mg/ml ), SEA (10 mg/ml ), oroccurs.Con A (2·5 mg/ml ) for 4 days, and subsequently supplementedwith 50 U/ml of interleukin-2 (IL-2) for another 3 days. These

MATERIALS AND METHODSsuperantigen and mitogen concentrations have been optimizedin pilot experiments. The cells were then collected, washed,Mice

Swiss mice, BALB/c, C57/BL, DBA/1, C3H.Q, C3H.NB, and stained with anti-Vb antibodies. All the samples wereanalysed with a fluorescence-activated cell sorter (FACSscan;C3H/Hej, NFR/N, NOD.Q (obtained by 10 backcrosses in

which the MHC gene region of B10.Q was inserted into the Becton Dickinson). The stained cells were gated using forwardand side scatter encompassing the blast population.non-obese diabetic (NOD) mouse), (B10.RIIIXRIIIS/J )F2,

congeneic mice B10M, B10Q, B10B and B10P were main-tained in the animal facility of the Department of Clinical

RESULTSImmunology, University of Goteborg, Goteborg, Swedenunder standard conditions of light and temperature. Both male Vb11+ T-cell expansion by TSST-1 displays different magnitude

in mice with different genetic backgroundand female mice were used as the source of splenocytes.

Splenocytes from different mouse strains were stimulated withReagents and culture medium

TSST-1 for 3 days. Subsequently, the cells were expanded withHighly purified TSST-1 and SEA were purchased from Toxin

IL-2 for another 4 days. The lymphoblasts recovered wereTechnology (Sarasota, FL). Concanavalin A (Con A) was

then examined for the expression of Vb11 antigen by flowbought from ICN Biochemicals (Cleveland, OH). Anti-mouse

cytometry. Table 1 summarizes the pattern of Vb11+ T-cellThy 1.2 antibody-coupled microbeads were bought from

expansion in response to TSST-1. The Vb11+ T cells fromMiltenyi Biotec GmbH, Bergisch Gladbach, Germany.

mice bearing MHC class II I-E molecules, including BALB/cPhycoerythrin (PE)-conjugated anti-GK1.5 (anti-CD4) and

(H-2d), C3H/Hej (H-2k), C3H.NB (H-2p), andfluorescein isothiocyanate (FITC)-conjugated anti-Lyt2 (anti-

(B10.RIIIXRIIIS/J )F2 (H-2r), did not expand upon stimula-CD8) were purchased from Becton Dickinson (Mountain

tion with TSST-1. In mice bearing I-A molecules alone, theView, CA). FITC-conjugated anti-immunoglobulin antibodies

Vb11+ T cells from the mice of an H-2q haplotype, includingwere bought from Dakopatts (Glostrup, Denmark). FITC-

Swiss, DBA/1, C3H.Q, NFR/N, and NOD.Q, were found toconjugated RR3-15 (anti-Vb11), MR5 (anti-Vb8.1,8.2), RR-7

be expanded by TSST-1. However, the Vb11+ T cells from(anti-Vb6) and PE-conjugated (anti-Vb3) were purchased from

C57/BL mice, being of an H-2b haplotype, did not expand inPharMingen (San Diego, CA). The culture medium con-

response to TSST-1. As controls, Vb60- or Vb8-bearing T cellssisted of Iscove’s (Gibco, Paisley, UK) supplemented with

did not respond to TSST-1 stimulation (data not shown).10% fetal calf serum, 2-mercaptoethanol 5×10−5 , 2 m of-glutamine, and 50 mg/ml of gentamicin.

TSST-1 expands Vb11+ T cells in B10 congeneic mice carryingH-2q and H-2f but not H-2b haplotypesMHC class II gene transfectants

L cells and lymphoma M120 transfected with H-2q genes wereTo exclude the influence of non-MHC genes in Vb11+ T-cell

prepared by transfecting the cells with the plasmids UB12expansion by TSST-1, we next examined the expression of

containing the Abq-gene and UB14 containing the Aaq-gene,as described previously.12 The expression of MHC class II

Table 1. Expression of Vb11 TCR on T lymphocytes from variousmolecules by these cells was checked by flow cytometry.mouse strains upon stimulation with TSST-1

Preparation of cells Vb11+T celsThe preparation of spleen mononuclear cells (MNC) was H-2 (%)performed as previously described.10 Purified T cells andantigen presenting cells (APC) were obtained by treatment Mouse strain A E Medium TSST-1of spleen MNC (1×107/ml ) with anti-Thy1.2. coupled

Swiss q/s – 5·2±1·8 19·3±13·0microbeads (Miltenyi, Biotec GmbH, Bergisch Gladbach,C3H/Hej k k 2·0±0·2 2·5±0·8Germany). The procedure was performed as recommended byC3H.Q q – 6·6±1·3 18·2±2·8the manufacturer. Two rounds of purification were performed.C3H.NB p p 3·1±1·1 2·5±1·2The purity of the T cells and APC was assessed by flowNFR/N q – 5·2±0·3 15·4±1·8cytometry after staining of the recovered cells with anti-CD4,(B10.RIIIXRIIIS/J )F2 r r 3·8±2·0 0·5±0·2

anti-CD8, and anti-immunoglobulin antibodies. The resulting BALB/c d d 3·5±2·7 1·3±0·2cell populations contained 90–95% of CD4+ and CD8+ T cells C57/BL b – 6·4±1·2 6·9±3·1in purified T population, and of immunoglobulin+ cells in DBA/1 q – 3·6±0·4 17·8±2·1APC population, respectively. APC from spleen MNC and NOD.Q q – 6·1±1·5 12·9±3·2MHC class II transfectants were incubated with mitomycin C(25 mg/ml ) (Sigma, St. Louis, MO) for 20 min at 37°. Results are expressed as mean±SD.

© 1998 Blackwell Science Ltd, Immunology, 94, 1–4

Vb11+ T-cell expansion by TSST-1 3

Table 3. TSST-1 triggered expression of Vb3 and Vb11 TCR onTable 2. TCR Vb expression of T lymphocytes after stimulation withTSST-1 in congeneic B10 mouse strains purified T lymphocytes from B10B and B10Q mice in presence of

different q gene expressing antigen presenting cellsPercentages of T cells expressing

Mouse Vb3 Vb11Cells Stimulus (%) (%)strain Stimulus Vb3 Vb6 Vb8 Vb11

Tb+APCq Medium 1·8±0·3 6·3±0·2B10B Medium 3·3 10·1 12·1 4·0(H-2b) TSST-1 25·7 3·5 7·2 6·1 Con A 2·2±0·8 5·0±2·2

TSST-1 16·1±0·6 9·7±0·4B10Q Medium 4·8 7·6 12·9 6·0(H-2q) TSST-1 16·4 0·9 1·8 13·3 SEA 10·8±0·9 23·7±2·1

Tb+FTq Medium 2·9±1·7 6·9±0·8B10M Medium 5·7 ND ND 2·5(H-2f ) TSST-1 19·1 ND ND 20·0 Con A 2·3±1·1 5·1±2·2

TSST-1 21·1±5·1 5·9±1·2B10P Medium 5·6 ND ND 2·1(H-2p) TSST-1 21·7 ND ND 4·9 SEA 10·2±4·4 19·6±4·2

Tb+M120q Medium 2·7±0·3 6·9±1·5Con A 3·44 6·57ND, not done.TSST-1 15·0±2·1 8·3±1·9SEA 10·0±4·8 28·8±0·2

Vb11 TCR reactive with TSST-1 on splenocytes of congeneic Tq+APCb Medium 2·0±0·4 7·4±1·3B10 mice with different H-2 haplotypes (Table 2). As expected, Con A 2·3±0·9 5·1±4·9the B10P mice with an H-2p (I-E+ strain) did not display TSST-1 22·2±3·3 14·1±2·4

SEA 4·6±0·9 24·1±1·4Vb11+ T-cell expansion by TSST-1. In I-E negative strains,Tq+FTq Medium 2·6±0·2 6·9±0·1TSST-1 expanded Vb11+ T cells in both B10Q (H-2q) and

Con A 2·4±0·5 4·9±6·3B10M (H-2f ) mice but not in B10B (H-2b) mice. As controls,TSST-1 19·5±3·0 14·5±3·3Vb3+, but not Vb6+ and Vb8+ T cells, expanded in responseSEA 6·2±3·3 33·9±3·3to TSST-1. The results from congeneic B10 mice further

Tq+M120q Medium 3·5±1·0 7·2±1·6confirmed that TSST-1 selectively expands Vb11+ T cells in Con A 2·5±0·2 7·1±0·1I-E negative mice with an H-2q but not H-2b haplotype. TSST-1 21·5±2·5 13·5±1·3

SEA 8·6±3·9 23·13

APC with q gene do not restore Vb11+ T-cell expansion byData are expressed as mean±SD.TSST-1 in mice with H-2b miceTb: purified T cells from B10B mice.Tq: purified T cells from B10Q mice.To examine if the failure of Vb11+ T-cell expansion by TSST-1APCb: antigen presenting cells from B10B mice.in mice with an H-2b haplotype is caused by deficient presen-APCq: antigen presenting cells from B10Q mice.tation from APC, purified T cells from B10B mice wereFTq: L cells transfected with I-Aq.stimulated with TSST-1 in the presence of different types ofM120q: lymphoma cell line transfected with I-Aq.APC with q gene. As shown in Table 3, APC from B10Q mice,

L cells and M120 lymphoma transfected with a q gene did notrestore the ability of Vb11+ T cell to expand in response to mammary tumour virus (Mtv) 8, 9, and 11.14,15 Thus, the

frequencies of mature Vb11+ T cells in peripheral lymphoidTSST-1 by purified T cells from B10B mice. Interestingly,APC from B10B mice did have capacity to present TSST-1 in organs are generally low in I-E-bearing mice.16 Furthermore,

the remaining of Vb11+ T cells might be functionally inacti-stimulation of Vb11+ T cells from B10Q mice. In addition,APC with q gene could present TSST-1 to Vb3+ T cells from vated during T-cell development in thymus.17 Therefore, it is

reasonable to hypothesize that TSST-1 stimulation is notB10B mice.powerful enough to trigger the expansion of Vb11+ T cell inmice bearing I-E molecules. This result is in line with the study

Vb11+ T cells can be expanded by SEA in H-2b miceshowing that SEA stimulates Vb11+ T cells in I-E negativestrains but not in I-E positive strains.6To determine if there is a quantitative difference regarding

Vb11 TCR density on T cells, the freshly obtained Vb11+ The intriguing finding in our study was that among theI-E negative strains Vb11+ T cells from mice with H-2q andT cells from B10Q and B10B mice were stained with anti-

Vb11 antibodies. The staining intensity of Vb11+ T cells in H-2f but not H-2b haplotypes responded to TSST-1 stimula-tion. There are two possible ways to explain the differentialB10Q and B10B mice are 52±12 and 48±7, respectively (NS).

Furthermore, SEA greatly expanded Vb11+ T cells from both magnitude of Vb11+ T-cell expansion by TSST-1 in H-2q ascompared to H-2b mice. It is established that a trimolecularB10Q (29·9%) and B10B (23·5%) mouse strains.complex consisting of an MHC class II molecule, the TCR,and a superantigen is essential for functional T-cell

DISCUSSIONresponses.1,18,19,20 Individual class II molecules vary in theireffectiveness at presenting superantigens to T cells and can beThe peripheral TCR repertoire is shaped through positive and

negative selection by interaction with self-peptides associ- ordered in a hierarchy. It has been shown a hierarchy for thepresentation of TSST-1, where H-2b<H-2d<H-2k.21 Theated with MHC molecules.13 It has been shown that T cells

expressing Vb11 TCR are partially deleted in the presence of superantigen presenting capacity of H-2q as compared withH-2b remains to be determined. In this respect, it could beI-E molecules and the gene products of endogenous mouse

© 1998 Blackwell Science Ltd, Immunology, 94, 1–4

Y.-X. Zhao et al.4

hypothesized that the difference with respect to the superanti- REFERENCESgen-presenting capacity between isotypic variations of MHC

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22. B T., L S., Y A., R C. & T A.We thank Dr R. D. Inman and Dr T. Trollmo for critical reading of (1991) Experimental Staphylococcus aureus arthritis in mice. Infectthe manuscript. This work was supported by grants from the Goteborg Immun 59, 2615.Medical Society, the Swedish Association against Rheumatism, the 23. Z Y.-X., A A., K T. & T A.King Gustaf ’s 80 Years Foundation, the Nanna Svartz Foundation, (1995) Overexpression of the T cell receptor Vb3 in transgenicthe Swedish Medical Research Council, the University of Goteborg, mice increases the mortality during infection by enterotoxin A

producing Staphylococcus aureus. Infect Immun 63, 4463.and the A.-G. Crafoord Foundation.

© 1998 Blackwell Science Ltd, Immunology, 94, 1–4