Auto-presentation of Staphylococcal enterotoxin A by mouse CD4<sup>+</sup> T cells

doi:10.4236/oji.2011.11002

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Vol.1, No.1, 8-14 (2011) Open Journal of Immunology

doi:10.4236/oji.2011.11002

Auto-presentation of Staphylococcal enterotoxin A by

mouse CD4+ T cells

—Auto-presentation by CD4+ T cells

Reuven Rasooly*, Paula M. Do, Bradley J. Hernlem

Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, USA; *Corresponding

Author: reuven.rasooly@ars.usda.gov

Received 27 May 2011, revised 12 June 2011, accepted 20 June 2011.

ABSTRACT

The currently accepted model for superantigen

(SAg) induced T cell activation suggests that

SAg, without being processed, cross link both

MHC class II, from Antigen Presenting Cells

(APC), and V-



, from T-cell receptor (TCR), initi-

ating nonspecific T-cell activation. This T-cell

proliferation induces a massive cy tokine release

associated with several human diseases. It is

thought that mu rine CD4 + T cells do not express

MHC class-II molecules. However, we discov-

ered that a subtype of mouse naïve CD4+ T cells

expresses MHC class II on their cell surface and

that these CD4+ T cells can perform the role of

both APC and T cells, able to present Staphy-

lococcal enterotoxin A (SEA) to itself or neigh-

boring CD4+ T cells via MHC class II, thus in-

ducing massive CD4+ T cell proliferation. Treat-

ment with neutralizing anti MHC class II anti-

body inhibits this CD4+ T cell proliferation re-

sponse. The fact that murine CD4+ T cells ex-

press MHC cl ass II offe rs new insight about S Ag

activity. Based on our findings, we propose re-

vising and extending previous models for SAg

induced T cell activation, altering previous mo-

dels of MHC class II restriction of T cell re-

sponses to SEA as well as the requirement for

SAg processing.

Keywords: MHC Class II; T-Cell; Enterotoxin

1. INTRODUCTION

Staphylococcus aureus is a major bacterial pathogen

causing several diseases including food-borne illnesses

[1]. S. aureus produces a grou p of 21 (known) staphylo-

coccal enterotoxins (SEs) that have two separate bio-

logical activities: they cause gastroenteritis in the gas-

trointestinal tract and act as a superantigen (SAg) caus-

ing massive activation and proliferation of T-cells and

cytokine release [1]. Several studies have shown that

there is a relation between emetic and superantigenic

activities [2,3]. SAg plays an important role in patho-

genesis by undermining the specificity of the adaptive

immune response. They are reported to be associated

with etiology of several human diseases including toxic

shock syndrome, Kawasaki disease, guttate psoriasis,

eczema, rheumatoid arthritis and scarlet fever [4].

The current model for superantigen activity suggests

that native, unprocessed SAg bind directly to the -

helical chain of the MHC class II, outside the peptide

binding groove of the antigen presenting cell (APC) [5].

This binding takes place without proteolytic degradation

and fragmentation, internalization, and re-expression of

the SAg degraded short fragments on the cell surface.

This native SAg is recognized and interacts with a large

number of T-cells, which all share particular sequences

within the variable region of the  chain (V-



) of the T

cell receptor (TCR), thereby stimulating ~20% of the

naïve T-cell population [6]. This trimolecular interaction

triggers massive proliferation of the T-cells subsets and

production of large quantity of cytokines that can have

pathological effects. The currently accepted model,

therefore, suggests that both types of cells; APC and T

cells, are needed for SAg induced T-cell proliferation

[7,8].

The currently acceptable view is that murine T-cells

do not express MHC class-II molecules [9-13], and that

professional APCs expressing MHC class II are required

for SAg induced T cell proliferation [7]. However, the

present study demonstrates for the first time that mouse

naïve CD4+ T cells



TCR express MHC class II on

their cell surface. Our data suggest that these T cells act

as APCs, capture staphylococcal enterotoxin A (SEA),

and present the SEA-MHC class II complex to itself or

R. Rasooly et al. / Open Journal of Immunology 1 (2011) 8-14

neighboring CD4+ T cells targeting their own surface

molecules. These events then cause proliferation. When

anti MHC class II antibodies are added to purified dou-

ble positiv e CD4+ T cells, they block T-cell auto presen-

tation and inhibit T cell proliferation.

2. MATERIAL AND M ETHODS

2.1. Chemicals and Reagents

SEA was obtained from Toxin Technology (Sarasota,

FL). Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU)

was obtained from Calbiochem (San Diego, CA). Phy-

tohemagglutinin (PHA) was obtained from Sigma Al-

drich (St Louis, MO). Anti-CD3 epsilon chain labeled

(PE), anti-



TCR labeled pacific blue, anti-CD4 anti-

body labeled with APC an ti-MHC class II an tib od y I-Ek,

anti MHC class II antibody against the subregion en-

coded glycoproteins I-Ab, I-Ad, I-Aq, I-Ed, I-Ek. And

anti-MHC class II antibody labeled with FITC or APC

were obtained fro m eBioscience (San Diego, CA). An ti-



3 T-cell receptor was obtained from BD Pharmingen

(Franklin Lakes, NJ). Imunomagnetic beads conjugated

with antibodies directed against CD3 and CD28 were

obtained from Invitrogen (Carlsbad, CA), as well as

CD4+ T-cell positive and negative isolation kits.

2.2. Splenocyte Isolation

Spleens from C57BL/6 female mice were aseptically

removed and disrupted using a syringe and needle in

Russ-10 cell culture medium (made by combining 450

ml of RPMI 1640 medium without glutamine (Gibco,

Carlsbad, CA), 50 ml Fetal bovine serum (Hyclone,

Logan, UT), 5 ml 200 mM glutamine (Gibco), 5 ml anti-

biotic-antimycotic (Gibco; containing penicillin, strep-

tomycin, and fungizone), 5 ml nonessential amino acid

mix (Gibco), 5 ml sodium pyruvate (Gibco), and 0.25 ml

of 100 mM beta mercaptoethanol (Sigma)). Cells were

centrifuged at 200 × g at 4˚C for 10 min. Red blood cells

were then lysed by adding 5 mL of red cell lysis buffer

(0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA).

Cells were again centrifuged and resuspended in Russ-10

medium, and viable cells were counted using trypan blue

and a hemocytometer.

2.3. Positive or Negative Isolation of Murine

CD4+ T Cells

Murine CD4 + T cells were isolated using either a posi-

tive (Dynabeads Mouse CD4 L3T4) or negative selec-

tion kit (Dynal Mouse CD4 Negative Isolation Kit), ac-

cording to the manufacturer’s instructions. Briefly, for

positive isolation, splenocytes were resuspended in iso-

lation buffer (PBS supplemented with 0.1% BSA and 2

mM EDTA) at a concentration of 1 × 107/mL, and in-

cubated with washed Dynabeads (25 l of Dynabeads

per 107 cells) for 20 mins on ice with gentle rotation.

After incubation the cells and Dynabeads were placed on

a magnet for 2 mins. The supernatant was removed and

the bead-bound cells wer e washed with isolation bu ffer 3

times. The bead-bound cells were resuspended in Russ-

10 media (107 cells per 100 l of media) and DE-

TACHaBEAD mouse CD4 was added (10 l per 107

cells) and incubated for 45 mins with gentle rotation at

room temperature. The detached cells were washed 3

times and resuspended in media. For negative isolation

of CD4 cells heat inactivated FBS and antibody were

added to splenocytes and incubated for 20 mins on ice.

The cells were washed with isolation buffer, and

pre-washed mouse depletion dynabeads were added and

incubated for 15 mins with gentle rotation at room tem-

perature. The cells and dynabeads were placed on a

magnet and the supernatant was obtained and washed.

The supernatant contained the negatively isolated mouse

CD4 T cells.

2.4. Superantigen Induced Naive T-Cell

Proliferation Assay

Cells were placed in 96-well plates (1 × 106/mL, 0.2

mL) in Russ-10 medium and treated with various con-

centrations of SEA ranging from 0.5 to 200 ng/ml fol-

lowing incubation at 37˚C in a 5% CO2 incubator. After

incubation for 48 h, cell proliferation was measured by

adding Bromodeoxyuridine (5-bromo-2-deoxyuridine,

BrdU), which was incorporated into the DNA of divid-

ing cells, 4 h before fixation as described by manufac-

turer instructions (Calbiochem, San Diego, CA). Spec-

troscopic measurements were made at an optical density

of 620 nm and 45 0 nm.

2.5. Flow Cytometry

One and a half million cells were placed through a

strainer and labeled with prospective antibodies for 40

mins. Cells were washed twice in 200 L of PBS and

resuspended in 0.5 mL of PBS. Flow cytometry and cell

sorting were performed using a FACS Vantage SE (Bec-

ton Dickinson) fitted with a Cobolt CalypsoTM 100 mW

491 nm laser (Cobolt AB, Sweden) and a cube 40 mW

640 nm laser (Coherent, Auburn, CA). The fluorescence

of FITC and PE labels were quantifed by excitation at

491 nm using 530/30 and 585/42 bandpass filters, re-

spectively. Fluorescence of APC was quantified by exci-

tation at 640 nm using a 676/29 bandpass filter (Sem-

rock, Rochester, NY).

2.6. Expansion of Cell Sorted Cells

After sorting cells were stimulated with immunomag-

netic beads coated with anti-CD3/28 in Russ-10 con-

R. Rasooly et al. / Open Journal of Immunology 1 (2011) 8-14

taining IL- 2 (30 U/mL).

2.7. Statistical Analysis

Statistical analysis was performed using SigmaStat 3.5

for Windows (Systat Software, San Jose, CA). Multiple

comparisons were made of PHA or with increasing con-

centrations of SEA that induce splenocytes or purified T

cell proliferation. The experiments were repeated at least

three times, and results with p < 0.05 were considered

statistically significant.

3. RESULTS

3.1. The Effect of APC-CD4+ T Cells Ratio on

Superantigen Induced Naive T-Cell

Proliferation

In order to activate CD4+ T-cells, a 10 to 1 ratio of

APCs to T cells is needed [14]. Our hypothesis is that

the ratio between APC and T cell is important for effi-

cient T-cell proliferation, and that there would be a re-

duction in CD4+ T-Cell proliferation response after al-

tering this ratio, because reduction in APCs limits the

number of CD4+ T cells that can bind to the SEA-APC

complex. To test this hypothesis, we increased CD4+ T

cell concentration by utilizing two types of CD4+ T cell

isolation methods; negative selection which increases the

concentration of CD4+ T cell from 22% in the average

spleen to 90%, or positive selection with an average pu-

rity of 95%. The isolated CD4+ T cells from a single

spleen preparation were cultured with three concentra-

tions of the superantigen SEA 0.5, 1 and 200 ng/ml.

Proliferation was measured on day 1 by BrdU incorpora-

tion. As shown in Figure 1, SEA induced proliferat i o n in

a dose dependent manner in all three cell cultures. Sur-

prisingly, the highly enriched positive isolated CD4+ T,

which presumably has the lowest concentration of APC

cell, provided the highest signal with highest signal-

to-noise ratio. These experiments raise the questions

whether depleting APC paradoxically increases prolife-

ration, and how complete elimination of APCs will ef-

fect proliferation.

3.2. The Superantigen Staphylococcal

Enterotoxin A Induces Proliferation

of CD4+ T Cells in the Absence of APC

To determine how elimination of APCs affects SEA

induced T cell proliferation, CD4+ T cells were purified

from splenocytes by positive and negative selection. In

these experiments, SEA was used at a concentration of

200 ng/ml. To determine the absence of APC, APC-de-

pendent mitogen PHA was used at concentration of 10

g/ml. Antibody against MHC class II was used to block

the interaction between MHC class II and the variable

region of the TCR. As shown in Figure 2, the APC-de-

splenocyteCD4 (pos)CD4 (neg)

OD 450 nm

0.0

0.1

0.2

0.3

0.4

0 ng/ml

0.5 ng/ml

1 ng/ml

200 ng/ml

Figure 1. SEA activation of purified CD4+ T-cells. Splenocytes,

positively selected, and negatively selected CD4+ T-cells were

incubated with increasing concentrations of SEA. After incu-

bation for 1 day newly synthesized DNA was measured. Error

bars represent standard errors and n = 3.

Figure 2. SEA induces proliferation of double purified CD4+ T

cells Splenocytes, positive selected and double positive se-

lected CD4+ T cells were incubated with SEA in the presence

or absence of antibody against MHC class II subregion glyco-

proteins I-Ab, I-Ad, I-Aq, I-Ed, I-Ek or I-Ek alone. After incuba-

tion for 2 days, newly synthesized DNA was measured. Error

bars represent standard errors and n = 3.

R. Rasooly et al. / Open Journal of Immunology 1 (2011) 8-14

pendent mitogen, PHA has high proliferative effect on

splenocytes, which contain large number of APCs. The

data also show a low effect on CD4+ T cells with posi-

tive isolation, and no proliferative effect on positive and

negative selection of CD4+ T cells. These observations

suggest that the double positive purified CD4+ T cells

lack APCs. This result is novel and interesting because

SEA was able to induce proliferation of purified CD4+ T

cells in the absence of MHC-class II expressed on APC.

However, anti MHC class II antibody against the subre-

gion encoded glycoproteins I-Ab, I-Ad, I-Aq, I-Ed and

I-Ek, but not I-Ek alone, blocked proliferation of the

doubled purified murine CD4+ T cells. This shows that

the SEA induced proliferation response is MHC class II

dependent. These observations suggest the presence of

MHC class II despite the lack of APCs.

3.3. Naïve CD4+ T Cells Can Express MHC

Class II

The presence of MHC class II in purified naïve CD4+

T cells without APC raises the question of the source of

the MHC class II molecules. Our hypothesis was that

Presort analysis of naïve splenocyte cells

(a) (b) (c) (d)

Post sort analysis of naïve splenocyte cells

(e) (f) (g) (h)

(i)

Figure 3. Naïve CD4+ T cells can express MHC class II Mouse splenocyte were immunostained with anti CD4, anti CD3, anti 

TCR and anti MHC class II antibody were simultaneously analyzed by flow cytometry in a single analysis. The data prior to sorting

show that 2.6% of the gated splenocytes are positive for all 4 antibodies (A-D). Cell sorting enrichment increased concentration of T

cells expressing MHC class II (E-H). (I) fluorescent microscopy of CD4+ T-cells, anti-CD4 antibody (is labeled with APC (red) and

anti-MHC class II is labeled with FITC (green). The data suggest that these are indeed CD4+ T cells expressing MHC class II.

R. Rasooly et al. / Open Journal of Immunology 1 (2011) 8-14

CD4+ T-cells become activated when they are presented

with SAg by MHC class II molecules that are expressed

on their surface. To test this hypothesis we labeled naïve

mouse splenocyte cells with anti CD4 fluorescein la-

beled (APC) antibody, anti MHC class II fluorescein

labeled (FITC), anti CD3 labeled (PE) and anti



TCR

labeled (pacific blue). These cells were analyzed by flow

cytometer FACS Calibur. Representative data from three

independent experiments show 22% of splenocytes are

CD3 and CD4 positive (Figure 3(a)). From this popula-

tion 3.1% are positive for CD3 and MHC class II, (Fig-

ure 3(b)). 4.2% are positive for CD4 and MHC class II

(Figure 3(c)). And 2.6% are positive for αβ TCR cells

and MHC class II (Figure 3(d)). Cell sorting increased

the concentration of this fluorescin labeled T cell sub-

type (Figure 3(e)-(h)). We also used confocal fluores-

cent microscopy (Figure 3(i)) and demonstrated that

these highly purified CD4+ T cells are double labeled

with anti-CD4 antibody (labeled with APC (red) and

anti-MHC class II is labeled with FITC (green), sug-

gesting these murine CD4+ T cells express MHC class II.

3.4. Expansion of CD4+ T Cells Expressing

MHC Class II

We performed an ex vivo assay that studied long term

expansion of this cell and demonstrated that isolated

CD4+ T cells expressing MHC class II can proliferate

without using autologous APCs, and can be expanded

with immunomagnetic beads conjugated with costimu-

latory signals anti-CD28 mAb and T cell activation anti-

CD3 mAb. Without this antibody the cells did not pro-

liferate. Our result also shows that the exponential pro-

liferation of this CD4 + T cell was maintained only for 13

days with an expansion rate of 40-fold (Figure 4(a)).

Flow cytometry indicated that during this expansion time

CD4+ T cells expressed high levels of MHC class II

molecule on their surface (Fi gure 4(b)).

4. DISCUSSION

Our studies demonstrate for the first time a subtype of

mouse naïve CD4+ T cells express MHC class II mole-

cules on the cell surface. We showed that SEA was able

to induce high T cell proliferation in dose dependent

manner on both negatively and positively selected mouse

naïve CD4+ T cells. The proliferative response of these

selected CD4+ T cell was similar to the response from

splenocytes. SEA was able to induce CD4+ T cell pro-

liferation even in the absence of APCs. This may suggest

that the safety mechanism requiring cellular interaction

between two different types of cells, accessory cell and T

cell does not occur in the response to SAg, consequently

inducing T cell over-activation with massive cytokine

release.

Treatment with neutralizing anti MHC class II anti-

body totally inhibits the proliferation of these CD4+ T

cells in the presence of SEA. These data suggest our

CD4+ T cells were able to present SEA via MHC class II

and provide sufficient accessory signals to themselves or

neighboring CD4+ T cells, triggering proliferation. Con-

sequently, they performed the roles of both professional

APCs and T cells. This may suggest that this T cell may

(a) (b)

Figure 4. Expansion of CD4+ T cell expressing MHC class II. Naïve CD4+ T cells expressing MHC class II molecule were iso-

lated from C57BL/6 mouse splenocytes by FACS Calibur. The purified Naïve CD4+ T cells were cultured in 96 well microplates.

At day 0, 7, 10, 13 and 17, cells were stimulated with anti-CD3/anti-CD28 mAb-coated beads and counted using the trypan blue

dye exclusion test to evaluate the fold increase in their numbers. Error bars represent standard errors and n = 3.

R. Rasooly et al. / Open Journal of Immunology 1 (2011) 8-14

Super-

antigen

T-Cell

APC

MHC





TCR

Antigen

PROLIFERATION

T-Cell

MHC





TCR

PROLIFERAT ION

MHC





PROLIFERATION

ORR

(a) (b)

Figure 5. The current model (a) and the proposed model (b in addition to a) explain how SEA induces T cell proliferation. The

current model suggests that SAg binds to two molecules on two separate cells; the V-



portion of the TCR and the MHC class

II on APC forms a bridge between the T cell and APC induces proliferation to large number of T cells. The proposed mecha-

nism suggests that T cells express both molecules; MHC class II and TCR. Therefore, activation by SAg may require only one

type of cell performing the role of both APC and T cell. SAg binds to both molecules in neighboring T cells or to both the

TCR and MHC class II on the same cell causing massive T cell proliferation.S

play a role as a sensor cell and first line of host defense

against S. aureus infection.

Our findings demonstrate that subtype of CD4+ T cells

express MHC class II and may act as professional APC,

process SAg and pr esen t th em to n e ighbo ring T cells and

induce CD4+ T cell proliferation even in the absence of

macrophages, dendritic cells or B lymphocytes. This ob-

servation may change the interpretation of earlier ex-

periments that used fixed APCs that are metabolically

incapable to uptake and process the superantigens, but

were able to present SEs and activate T cells. Therefore,

the question as to whether processing of SEs is not re-

quired was not addresse d by t he pre vi ous st u di es.

White et al. [15] and Fleischer et al. [16] demon-

strated that T cell response to SEA are not MHC class II

restricted and that mouse TCR can recognize non host

APC expressing MHC class II alleles from different spe-

cies. However, these authors did not tak e into considera-

tion that CD4+ T cells that express MHC class II mole-

cules on their cell surface can act as APCs. Therefore,

their experimental data cannot evaluate whether MHC

restriction is violated in response to SEs.

In conclusion the paradigm (Figure 5(a)) that sug-

gests the only possible mechanism that SAg induces T

cell proliferation is by binding to two separate cells,

APC and T-cell, may be need to be revised. Our revised

model (Figure 5(b)) suggests that alongside the above

mechanism SAg may be able to induce T cell prolifera-

tion by binding to only one type of CD4+ T-cell express-

ing MHC class II. Performing the role of both accessory

cell and T cell, these cells present SAg via MHC class II

to themselves or neighboring CD4+ T-cells, triggering

nonspecific massive T-cell proliferation. The revised

model offers further insight into the in vivo mechanism

of SAg activity.

5. ACKNOWLEDGEMENTS

We thank Anne Bates for her help with confocal microscopy and

Daphne Tamar and Sharon Abigail for helpful discussions.

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