Superantigen-presentation by rat major histocompatibility complex class II molecules RT1.B l and...

Superantigen-presentation by rat major histocompatibility complexclass II molecules RT1.Bl and RT1.Dl

Henry Dlaske,1 Hatice Karauzum,1

Elisa Monzon-Casanova,1 Ronald

Rudolf,1 Lisa Starick,1 Ingrid

Muller,1 Gerhild Wildner,2 Maria

Diedrichs-Mohring,2 Norbert

Koch,3 Tohru Miyoshi-Akiyama,4

Takehiko Uchiyama,4 Kurt

Wonigeit,5 Bernhard Fleischer,6

Silke Overbeck,7 Lothar Rink7 and

Thomas Herrmann1

1Institute for Virology and Immunobiology,

Julius-Maximilians-University Wurzburg,

Wurzburg, 2Section of Immunobiology,

Department of Ophthalmology, Ludwig-

Maximilians-University, Munich, 3Division of

Immunobiology, Institute of Genetics,

University of Bonn, Bonn, Germany,4Department of Microbiology and Immunology,

Tokyo Womens’s Medical University School of

Medicine, Tokyo, Japan, 5Transplantation

Laboratory, Department of Visceral and

Transplantation Surgery, Medical School

Hannover, Hannover, 6Bernhardt Nocht

Institute for Tropical Medicine, Hamburg, and7Institute of Immunology, University Hospital,

RWTH Aachen, Germany

doi:10.1111/j.1365-2567.2008.03033.x

Received 11 June 2008; revised date 9

November 2008, 1 December 2008; accepted

2 December 2008.

Correspondence: Dr T. Herrmann, Institut

fur Virologie und Immunbiologie, Julius-

Maximilians Universitat Wurzburg,

Versbacherstrasse 7, D-97078 Wurzburg,

Germany. Email: herrmann-t@vim.

uni-wuerzburg.de

Senior author: Thomas Herrmann

Summary

Rat major histocompatibility complex (MHC) class II molecules RT1.Bl

(DQ-like) and RT1.Dl (DR-like) were cloned from the LEW strain using

reverse transcription–polymerase chain reaction and expressed in mouse

L929 cells. The transduced lines bound MHC class II-specific monoclonal

antibodies in an MHC-isotype-specific manner and presented peptide

antigens and superantigens to T-cell hybridomas. The T-cell-hybridomas

responded well to all superantigens presented by human MHC class II,

whereas the response varied considerably with rat MHC class II-trans-

duced lines as presenters. The T-cell hybridomas responded to the pyro-

genic superantigens Staphylococcus enterotoxin B (SEB), SEC1, SEC2 and

SEC3 only at high concentrations with RT1.Bl-transduced and RT1.Dl-

transduced cells as presenters. The same was true for streptococcal pyro-

genic exotoxin A (SPEA), but this was presented only by RT1.Bl and not

by RT1.Dl. SPEC was recognized only if presented by human MHC class

II. Presentation of Yersinia pseudotuberculosis superantigen (YPM)

showed no MHC isotype preference, while Mycoplasma arthritidis super-

antigen (MAS or MAM) was presented by RT1.Dl but not by RT1.Bl.

Interestingly, and in contrast to RT1.Bl, the RT1.Dl completely failed to

present SEA and toxic shock syndrome toxin 1 even after transduction of

invariant chain (CD74) or expression in other cell types such as the sur-

face MHC class II-negative mouse B-cell lymphoma (M12.4.1.C3). We dis-

cuss the idea that a lack of SEA presentation may not be a general feature

of RT1.D molecules but could be a consequence of RT1.Dl b-chain allele-

specific substitutions (arginine 80 to lysine, asparagine 82 to aspartic acid)

in the extremely conserved region flanking the Zn2+-binding histidine 81,

which is crucial for high-affinity SEA-binding.

Keywords: invariant chain; major histocompatibility complex class II;

major histocompatibility complex–peptide interaction; superantigens

Please cite this article in press as: Dlaske H. et al. Superantigen-presentation by rat major histocompatibility complex class II molecules RT1.Bl

and RT1.D1, Immunology (2009) doi: 10.1111/j.1365-2567.2008.03033.x

Abbreviations: CD, cluster of differentiation; CDR, complementarity determining region; DR, HLA-DR; DaMIg, donkey antimouse immunoglobulin; DQ, HLA-DQ; GFP, green fluorescent protein; gpMBP, guinea pig myelin basic protein; HLA, humanleucocyte antigen; MAS, Mycoplasma arthritidis superantigen; MHC, major histocompatibility complex; SAg, superantigen; SE,staphylococcal enterotoxin; SPE, streptococcal pyrogenic exotoxin; TCR, T-cell receptor; TSST-1, toxic shock syndrome toxin 1;YPM, Yersinia pseudotuberculosis mitogen.

e572 � 2009 Blackwell Publishing Ltd, Immunology, 128, e572–e581

I M M U N O L O G Y O R I G I N A L A R T I C L E

Introduction

Superantigens (SAgs) are microbial products, which acti-

vate T cells by simultaneous binding to T-cell receptor

(TCR) and major histocompatibility complex (MHC)

class II molecules. Most SAgs bind predominantly to Vb-

encoded parts of the TCR, a mode of interaction that

results in a high frequency of activated T cells expressing

TCR with certain SAg-reactive Vb domains, which even-

tually may contribute to numerous pathological condi-

tions.1–3 Most SAgs have been reported to activate T cells

expressing homologous Vb TCR groups in mouse, rat

and human4–7 and can be presented by xenogeneic MHC

molecules. The MHC class II-binding and presentation of

some SAgs has been very well characterized for human

and mouse molecules, while very little is known about

their presentation by rat MHC class II molecules. Such

knowledge would be desirable, because rats provide exper-

imental models for many pathological conditions includ-

ing autoimmune diseases, in which viral or bacterial SAgs

may be involved. Therefore, and to promote a better

understanding of SAg action and rat MHC function in

general, RT1.Bl (an HLA-DQ homologue) and RT1.Dl (an

HLA-DR homologue) from LEW rats have been cloned

and expressed. The analysis of these RT1.B and RT1.D

gene products is especially useful because they are identical

in l and l-variant RT1 (rat MHC) haplotypes and are

shared by the widely used strains LEW (Lewis), F344

(Fischer) and WKY (Wistar Kyoto). Furthermore, LEW is

a widely used model of organ-specific autoimmune dis-

eases such as experimental autoimmune encephalomyelitis,

uveitis or neuritis.

With respect to their MHC- and TCR-binding modes

SAgs can be divided into several groups. Based on crystal-

lographic results Fraser and Proft8 classified the SAgs

from the human pathogens Staphylococcus aureus or

Streptococcus pyogenes as follows: (i) binding to MHC

class II a-chain entirely peripheral to the bound antigen

peptide (peptide-independent binding), [this group

includes staphylococcus enterotoxin B (SEB) and the

highly homologous SEC1, SEC2, SEC3 and streptococcal

pyrogenic exotoxin A (SPEA)]; (ii) binding to MHC

a-chain and extension over the bound peptide (peptide-

dependent binding) as in the case of toxic shock

syndrome toxin 1 (TSST-1); (iii) zinc-mediated binding

to the b-chain and extension over the bound peptide,

such as in the case of SPEC; and (iv) SAgs that combine

binding modes (i) and (ii) and that can cross-link MHC

molecules such as SEA. Not included in this list are two

SAgs from Gram-negative organisms, which show no

homology to each other or to those SAg listed above.

One is the Mycoplasma arthritidis SAg (MAS), which is

often referred to as M. arthritidis mitogen or MAM; MAS

is produced by M. arthritidis, which was originally

isolated from an arthritic rat and promotes arthritis in

rats.9,10 The structural analysis of a co-crystal from MHC

class II, SAg and TCR revealed a unique mode of MHC

binding that prevented direct contact of MHC and TCR

and generated direct contact between the SAg and TCR

b-chain and a-chain.11 The other SAg is Yersinia

pseudotuberculosis mitogen (YPM), a virulence factor of

Y. pseudotuberculosis.12 The crystal structure of YPM is

available,13 but very little is known about its interaction with

TCR and MHC class II molecule, because no clear results

were obtained from directed mutation studies of YPM.14

Most SAgs have been isolated from the human pathogens

S. aureus and S. pyogenes and are much more potent in

humans than in mice, which to some extent reflects their

better binding to human (human leucocyte antigen; HLA)

than to mouse (H2) MHC class II molecules. With respect

to binding to certain MHC isotypes or alleles, better bind-

ing has been reported for SEA, SEB and SEC1 to DR and

for SEC3 and SPEA to DQ, but polymorphism within one

MHC class II isotype strongly affects these prefer-

ences.1,2,15–20 For TSST-1, binding to DR and DQ has been

reported in humans, and preferential binding to H2-A

compared with H2-E has been recorded for mice.17,21 In

most cases, SEA binds DR better than DQ,17,22 but shows a

preference for H2-A in mice.21,22 MAS binds to human DR

and DQ alleles, whereas the a-chain of H2-E enhances

binding considerably in mice and in chimeric mouse–

human MHC class II molecules.23,24 Information on MHC

isotype or allele preferences in SAg presentation by rat

MHC class II are not available, but it is known that HLA-

DR-transduced L929 cells can reconstitute the poor T-cell

response to SEB and SEC in rats.25

In this study RT1.Bl, RT1.Dl and rat CD74 were cloned

and expressed in mouse cells, which were than tested for

presentation of peptide antigens, binding of class II-spe-

cific antibodies and presentation of a wide range of SAgs

with highly divergent or not yet characterized MHC class

II binding properties. We tested the presentation of SAgs

to T-cell hybridomas, which allowed us to assess the rela-

tive SAg-presenting capacity of the cloned rat MHC class

II molecules and revealed a lack of SEA and TSST-1

presentation by RT1.Dl, which is discussed with respect

to current models of MHC–SAg binding.

Materials and methods

Animals

Six- to twelve-week-old (LEW/Crl) rats were provided by

the animal facility of the Institute for Virology and

Immunobiology (Wurzburg, Germany). Breeding pairs

were obtained from Charles River (Sulzfeld, Germany).

Rat strains LEW.1R14/Won and LEW/Ztm used to gener-

ate monoclonal antibodies (mAbs) HT11 and HT12 were

maintained in the animal facility of the Medizinische

Hochschule Hannover.

� 2009 Blackwell Publishing Ltd, Immunology, 128, e572–e581 e573

Superantigen-presentation by rat MHC class II

Antibodies and flow cytometry

Names and references for MHC class II mAbs are given in

Table 1 in the Results section. Purified mAbs and phycoer-

ythrin-labelled mAbs 14-4-4S, OX6 and OX17 were

obtained from (BD Biosciences, Pharmingen, Heidelberg,

Germany). The other MHC class II-specific mouse mAbs

were culture supernatants or ascites fluid from the respec-

tive hybridoma lines. The hybridomas producing the mAbs

HT11 and HT12 were generated from a LEW.1R14 rat after

immunization with two consecutive grafts of LEW skin

and four injections of LEW spleen and lymph node cells

using the same methods as previously described.26 Phyco-

erythrin- or fluorescein isothiocyanate-conjugated donkey

anti-mouse immunoglobulin or anti-rat immunoglobulin

was purchased from Dianova (Hamburg, Germany) and

normal mouse immunoglobulin was purchased from

Sigma-Aldrich (Deisenhofen, Germany). L180/1 and V65

served as isotype-matched controls. Staining was per-

formed as described elsewhere25,27 and cells were analysed

by two-colour immunofluorescence on a FACScan flow

cytometer using CELL QUEST software (Becton Dickinson,

Sunnyvale, CA). Staining with MAS–green fluorescent pro-

tein (GFP) and fluorescence-activated cell sorting (FACS)

analysis of transfected L929 cell lines and P3/2+CD80 cells

was performed as previously described.28 Cell sorting was

performed with a FACSVantage Cell Sorter (Becton Dick-

inson). Light scatter gates were set to include all viable

nucleated cells.

Bacterial SAgs and T-cell antigens

The SEB and SECs were obtained from Toxin Technology

(Sarasota, FL); MAS was purified as described

previously.29 Generation and purification of MAS–GFP

has been described elsewhere.28,30 Both SPEA and SPEC

were produced and purified as described in ref. 31 and

MAS, SEA and TSST-1 were produced as described in ref.

30. The YPM was purified as a recombinant product from

Escherichia coli XL1-blue carrying pQE30-6-hisypm by

using Ni–NTA agarose, as reported previously.32,33 Alpha

S2-casein from bovine milk was purchased from Sigma-

Aldrich, and guinea-pig myelin basic protein (gpMBP)

was a kind gift from Prof. Dr R. Gold (Department of

Neurology, Clinical Research Group for Multiple Sclero-

sis, Wurzburg, Germany).

Cell lines

The T-cell hybridoma 53/4 recognizes guinea-pig MBP

and its 68–88 peptide. It expresses the same Vb8.2 com-

prising TCR b-chain and was generated in the same

fusion as the previously characterized T-cell hybridoma

35/1.4 It recognizes a wide range of partially very distinct

SAgs (SEB, SEC1, SEC2, SEC3, SPEA, MAS, YPM). The

T-cell hybridoma 19 was generated as previously

described for the T-cell hybridoma 35/1,4 but using a

casein-specific T-cell line as fusion partner. This line has

been previously shown to be RT1.Bl-restricted as well as

RT1.Dl-restricted, defined by inhibition experiments with

respective MHC class II-specific mAbs.34 FRN2.7 is a

mouse T-cell hybridoma that expresses a transfected

human Vb2 TCR with reactivity to TSST-135 and SPEC,36

while SEA presentation was revealed with the ovalbumin-

specific mouse T-cell hybridoma 3DO54.8.37 P3/2 cells

are mouse DAP-3 fibroblasts expressing DR115 and were

kindly provided by Dr R.-P. Sekaly (Clinical Research

Institute, Montreal, Canada). P3/2+CD80 (DR1- and rat

Table 1. Binding of monoclonal antibodies to L929 transductants

Binding to

mAb Reported specificity References L929 RT1.Bl-L929 RT1.Dl-L929

10-2.16 H2-Ak,f,r,s,u 50 ) ++ to +++ )K25-137 H2-Ak,b,j,s,r,q,f 50 ) + to ++ )H150-13 H2-Ak,f,r,s 50 ) +++ )K22-203 H2-Ak,j,s,r,f 50 ) ++ )K25-8.7 H2-Ak,b,j,s,r,q,f 50 ) + to ++ )OX6 H2-Ab/RT1.Bb (all strains except BDII) 51 ) +++ )OX17 H2-E/RT1.Da-chain 51 ) ) +++

14-4-4S H2-Eak,d,p,r,u,v (Ia.7) rat MHC classII 52 ) ) +++

13.4 H2-Eak,d,p,r,u,v (Ia.7) 53 ) ) +++

41-A H2-Ek,d,p,r,u,v 54 ) ) ++

HT11 LEW.1R14 anti LEW This paper Negative Positive Negative

HT12 LEW.1R14 anti LEW This paper Negative Negative Positive

Binding strength of the mouse monoclonal antibody (mAb) was assessed by calculating the ratio of [mean fluorescence intensity (MFI) of anti-

body staining ) MFI of background staining]/(MFI of positive control staining ) MFI of background staining). As a positive control for RT1.Bl-

transduced cells we used OX6 mAb, for RT1.Dl-transduced cells OX17 was used. Degree of relative staining intensity is rated as: +, MFI 5–20%

of positive control; ++, MFI 21–50% of positive control; +++, MFI > 50% of positive control.


H. Dlaske et al.

CD80-positive) were generated by retroviral transduction

of rat CD8038 into P3/2 cells. M12.4.C3 is a BALB/c

mouse B-cell lymphoma with mutated Ad and Ed

b-chains and a defect in H2-A and H2-E surface expres-

sion.39 They were kindly provided by Dr L. Glimcher

(Harvard School of Public Health, Boston, MA). RAJI is a

human B-cell lymphoma and was provided by Dr R.

Accolla (University of Insubria, Varese, Italy).

Gene cloning and gene transduction

RT1.Bl expression vectors have been previously

described.4 RT1.Dl a- and b-chain genes were generated

by inserting reverse transcription–polymerase chain reac-

tion (RT-PCR) products from LEW rat lipopolysaccharide

blasts into the NcoI and BamHI sites of the retroviral

expression vector SFG S65T.40 Primer sequences were:

RT1.Dl a: forward 50-CCCATGCCATGGCCACCATTG

GAG-30, reverse 50-CCCGCGGATCCTCACAGGGCTCC

TTG-30; RT1.Dl b: forward 50-CCCATGCCATGGTGT

GGCTCGCC-30, reverse 50-CCCGCGGATCCTCAGTTCA

GGAGTCC-30. RatCD74 was inserted into to the EcoRI

and BamHI sites of pczCGZIEGZ vector, a bicistronic

vector with enhanced GFP as reporter.41 Primer sequences

for amplification were: forward 50-CTTCGAATTCGCCG

CCACCATGGATGACC AGCGCGACC-30, reverse 50-ACC

AGGATCCTCACAAGAACATTTGGCCCATA-30. Retro-

viral transduction of MHC class II genes and CD74 was

performed as described previously.4 Transduced cells were

selected by FACS sorting on a FACSvantage sorter.

Cell culture and SAg-mediated stimulation

Cells were cultured at 37� in 5% CO2 and 100% humidi-

fied atmosphere in RPMI-1640, supplemented as

described elsewhere.42 Single cell suspensions of lymph

nodes and splenocytes were generated by mincing

through a steel sieve. Nylon wool purification for enrich-

ment of T cells was performed.43 Stimulations were car-

ried out as previously described25 with some minor

modifications. For T-cell hybridoma stimulation, two

experimental protocols were used to test the transduced

fibroblast lines. For protocol A fibroblast lines (trans-

duced or non-transduced L929 cells or DAP3 cells) were

seeded at 2�5 · 104 cells each in 96-well, flat-bottom

plates. After one night, 5 · 104 T-cell hybridoma 53/4

cells and the respective SAgs were added to a final volume

of 200 ll. For protocol B 2�5 · 104 freshly detached fibro-

blast line cells and 5 · 104 T-cell hybridoma cells were

cocultured for 40 hr in 96-well, flat-bottom plates in

200 ll medium including SAg. Presentation by trans-

duced M12.4.1.C3 cells or RAJI cells was tested in

18–24 hr cultures using round-bottom plates. Peptide-

antigen-specific responses were measured using protocol

B for transduced fibroblasts and using 48-hr cultures in

round-bottom plates with 1 · 106 LEW rat thymocytes as

antigen-presenting cells. The concentration of mouse

interleukin-2 (IL-2) secreted by the T-cell hybridomas

was determined with the OptEIA-IL2-ELISA-Kit (BD

Biosciences Pharmingen). Samples were diluted with

culture medium when IL-2 content was above the range

of the assay (optical density > 2).

Results

Cloning and expression of RT1.Bl/RT1.Dl in L929cells

Retroviral expression vectors for the a- and b-chains of

RT1.Bl and RT1.Dl were generated as described in the

Materials and methods and used to express the rat MHC

class II molecules in mouse fibroblast L929 cells because

these cells do not express endogenous H2-A/E and are

capable of processing exogenous proteins.44 The sequence

of the RT-PCR products covered the entire coding

sequence. The RT1.D a sequence (GenBank: AAR87772.1)

obtained was identical to subsequently submitted RT1.D1

a sequences of the RT1l haplotype (for LEW AAV40645.1

and for F344 AAV40644.1).45 Our own and published

RT1.Dl b sequences45 were also identical. RT1.Bl-positive

and RT1.Dl-positive cells were enriched by FACSorting

and respective rat MHC class II expression was deter-

mined by flow cytometry (Fig. 1). Transfected cells

80 (a)

(b)

(c)

(d)

70

60 M1 M1

M1 M1

30

RT1B

Transductants splenocytes

RT1D

20

10

0

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40 Cou

nts

Cou

nts

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nts

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40

Cou

nts

30

20

10

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FL2-H FL2-H 100 101 102 103 104 100 101 102 103 104

FL2-H FL2-H 100 101 102 103 104 100 101 102 103 104

Figure 1. Flow cytometry of RT1.Bl-transduced (a) or RT1.Dl-trans-

duced (b) L929 cells and of LEW splenocytes (c and d). Staining

with phycoerythrin-conjugated OX6 (anti-RT1.B) and 14-4-4S (anti-

RT1.D) is depicted as open histograms. Staining with the isotype-

matched control antibody is depicted as filled histogram.



stained with similar intensity for the respective class II

molecule, whereas no signal could be detected in non-

transfected cells and for the non-transferred isotype. When

the mean fluorescence intensities of positive cells (gated in

M1) were compared with that of LEW rat splenocytes,

expression of both molecules on transduced cells was six

or seven times weaker than on the splenocytes.

Determination of MHC isotype specificity andcross-reactivity of rat and mouse MHC class II-specific mAbs

We tested the MHC class II expression of L929 cells,

native as well as transduced with rat MHC class II, with

various antibodies specific for mouse class II antigens and

rat RT1.B and RT1.D (Table 1). Binding of mouse anti-

bodies was revealed by phycoerythrin-labelled anti-mouse

immunoglobulin, and binding of the newly generated rat

allospecific mAbs HT11 and HT12 was visualized with

fluorescein isothiocyanate-labelled anti-rat immunoglobu-

lin. The mouse antibodies which were originally generated

against allogeneic mouse MHC class II molecules reacted

with the homologous rat MHC class II isotype (H2-

A:RT1B and H2-E:RT1D). The allospecific rat antibodies

HT11 and HT12 bound to RT1Bl and RT1Dl, respec-

tively.

Antigen and SAg presentation to rat T-cellhybridomas by L929 transductants

The suitability of the MHC class II transductants to

determine the restriction element of a given rat TCR was

tested with the help of T-cell hybridomas, which were

generated from rat CD4 cell lines with presumed RT1.Bl

or RT1.Dl restriction. The T-cell hybridoma 53/4

expresses a well-characterized TCR typical for gpMBP-

specific CD4 T cells, as they are found in LEW rats fol-

lowing experimental autoimmune encephalomyelitis

induction. Hybridoma 19 was generated from a casein-

specific CD4-positive LEW rat T-cell line that, based on

antibody inhibition data, was RT1.Bl-restricted as well as

RT1.Dl-restricted.34 As shown in Fig. 2, hybridomas 53/4

and 19 were stimulated with their cognate antigens pre-

sented by thymocytes, which are a common source of

antigen-presenting cells for rat T-cell line culture, RT1.Bl

or RT1.Dl transductants, respectively. The hybridoma 53/

4 responded only when RT1.Bl-transduced cells presented

the antigen, while hybridoma 19 responded only to anti-

gen presented by RT1.Dl transductants. Hybridoma 19

required approximately 10-fold more antigen than

hybridoma 53/4, because the only available preparation of

casein is a mixture of several bovine milk casein proteins.

Interestingly, the dose responses of hybridoma 19 to anti-

gen presented by thymocytes or transduced L929 cells

were similar, whereas 53/4 was 10- to 100-fold more

sensitive to gpMBP presented by thymocytes than to anti-

gen presented by L929 transductants.

Next, we tested the presentation of various SAgs by

transduced L929 cell lines to the LEW Vb8.2 TCR-posi-

tive T-cell hybridoma 53/4 (Fig. 3). P3/2+CD80 cells

(DR1-transduced mouse DAP-3 fibroblasts, which were

supertransduced with rat CD80) were used as the positive

control for the functionality of the test system because all

bacterial SAgs tested are known to be efficiently presented

by human MHC class II. The overall degree of stimula-

tion varied considerably between experiments so the

results were standardized by setting the average of stimu-

lation with the highest concentration of SAg presented by

DR-transduced cells as 100%. As expected, for the staphy-

lococcal enterotoxins, the T-cell hybridoma reaction was

strongest in the presence of DR1 with a detectable

response already at the second lowest dilution, achieving

10- to 20-fold higher levels of IL-2 compared with rat

MHC class II transductants. Interestingly, SEB-induced

IL-2 production could also be demonstrated in the pres-

ence of native L929 cells showing a comparable course of

IL-2 secretion, but with about half the amount obtained

with SEB presentation by rat MHC class II. The SEB

alone did not activate hybridoma 53/4 cells (data not

shown). The response to SEC2 is not shown in the graph,

but was almost identical to that elicited by SEC1 and

0

0 0·1 1 10 100 1000

Concentration of casein (µg/ml)

250

500

750

1000 Control

RT1B

RT1D

Thymocytes

IL-2

pro

duct

ion

(pg/

ml)

0·01 Concentration of gpMBP (µg/ml)

0·1 1 10 100

500

IL-2

pro

duct

ion

(pg/

ml)

1000

1500

2000 (a)

(b)

Figure 2. Interleukin-2 (IL-2) release of T-cell hybridoma 53/4 (a) or

T-cell hybridoma 19 (b) after 40 hr culture with indicated amounts

of guinea-pig myelin basic protein (gpMBP) (a) or casein (b): LEW

rat thymocytes (square), L929 untransduced (circle), L929 RT1.Bl-

transduced (triangle), L929 RT1.Dl-transduced (inverted triangle).


H. Dlaske et al.

SEC3. A response to SPEA could only be generated at

rather high SAg concentrations and was considerably

stronger with the HLA-DR-transduced cells than with

RT1Bl transductants as presenters, while no response was

seen with untransduced or RT1Dl transduced cells. The

T-cell hybridoma responded similarly well to MAS when

presented by RT1.Dl or DR1 while no response was seen

with RT1.Bl-expressing cells as presenters. These results

correlate well with the binding of MAS–GFP to RT1.Dl

transductants but not to RT1.Bl transductants (data not

shown). The YPM response showed no clear preference

for one of the rat MHC class II isotypes, while DR1 trans-

fectants elicited a drastically higher response. SPEA-

specific IL-2 production was poor with RT1.Bl transduc-

tants as presenters and completely absent with RT1.Dl or

untransduced L929 cells as presenters.

In summary, with the exception of MAS, the response

to all SAgs tested so far was much stronger with HLA-

DR1 transductants than with rat MHC class II transduc-

tants as presenters and only MAS and SPEA showed a

clear preference for an MHC class II isotype as the

presenting molecule.

Stimulation with SEA, TSST-1 and SPEC by varioustypes of RT1.Bl- and RT1.Dl-transduced cell lines

In the next set of experiments, we analysed the presenta-

tion of SEA, TSST-1 and SPEC by rat versus human

MHC class II molecules using the cell lines 3DO54.8

(SEA-reactive) and FRN2.7 (TSST-1-reactive and SPEC-

reactive) as responders. As referred to in the Introduction,

crystal structures demonstrated that all three SAg interact

with the MHC-bound peptide upon MHC binding. The

MHC class II binding of SEA and TSST-1 has also been

reported to differ strikingly depending on the presenting

cell type and expression of invariant chain or DM. In the

case of DR1-transduced HeLa cells SEA binding could

only be achieved after transduction of the invariant

chain.46 Therefore, the response to SAg was tested to the

SAg before and after transduction of rat invariant chain

(CD74), and with MHC class II-transduced M12.4.1.C3 B

lymphoma cells as presenters.

First, RT1.Bl- and RT1.Dl-transduced L929 cells were

further transduced with rat invariant chain expressed by a

biscistronic retroviral vector using enhanced GFP as the

reporter gene. Figure 4 shows that upon transduction of

the CD74 construct, expression of both RT1.Bl and

RT1.Dl increased, although the increase was rather weak

for RT1.Dl. Figure 5 shows two experiments of the T-cell

response to SEA and TSST-1 for CD74-negative and

CD74-positive rat MHC class II transductants in compar-

ison to RAJI cells and DR1-transduced fibroblasts. In

both cases, the SAg response was seen with RT1.Bl trans-

ductants but not with the RT1.Dl transductants and the

SEA response to DR1-transduced cells was substantially

better than to RT1.Bl transductants. The higher IL-2 pro-

duction seen in the experiments with the CD74 transduc-

tants may reflect the prolonged culture of 40 hr rather

120 (a)

(e)

(b)

(f)

(c)

(g)

(d)

(h)

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5

10

15 ControlRT1BRT1DHLA-DR1

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30

40

50

60

10

120

100

80

60

40

20

120

100

80

60

40

20

120

100

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60

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20

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100

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60

40

20

0 0·001 0·01 0·1 1 10 100

0 0·001 0·01 0·1 1 10 100

0 0·001 0·01 0·1 1 10 100

0 0·01 0·1 1 10 100

0 0·001 0·01 0·1 1 10

0 0·001 0·01 0·1 1 10 100

0 0·001 0·01 0·1 1 10 100

0 0·001 0·01 0·1 1 10 100

Concentration of SEB (µg/ml)

Concentration of SEC3 (µg/ml) Concentration of SPEA (µg/ml) Concentration of YPM (µg/ml) Concentration of MAS (ng/ml)

Concentration of SEB (µg/ml) Concentration of SEC1 (µg/ml) Concentration of SEC1 (µg/ml)

IL-2

pro

duct

ion

(%)

IL-2

pro

duct

ion

(%)

Figure 3. Interleukin-2 (IL-2) release by T-cell hybridoma 53/4 after 24–40 hr culture with the indicated amount of superantigen (a) staphylococ-

cal enterotoxin B (SEB), (b) SEB, (c) SEC1, (d) SEC1, (e) SEC3, (f) staphylococcal pyrogenic exotoxin A (SPEA), (g) Yersinia pseudotuberculosis

mitogen (YPM), (h) Mycoplasma arthritidis superantigen (MAS). (b) Depicts data from the SEB stimulation shown in (a) without the respective

data generated with DR1-transduced presenting cells. (d) Depicts data from the SEC1 stimulation shown in (c) without the respective data gener-

ated with DR1-transduced presenting cells. Superantigen (SAg) presenting cells were: DR1 transductant P3/2 CD80 (square), L929 untransduced

(circle), L929 RT1.Bl-transduced (triangle), L929 RT1.Dl-transduced (inverted triangle). Results were normalized for IL-2 production in the pres-

ence of DR1 transductants and the highest concentration of the respective superantigens. The average of three to five experiments is shown. Error

bars indicate standard deviation.



than 20 hr and increased surface expression of the RT1.Bl

molecule (Fig. 4) rather than a more efficient presentation

of the SAg by RT1.Bl in the presence of CD74.

Figure 6 shows a comparison of the response to SEA,

TSST-1 and SPEC presented by RT1.Bl or RT1.Dl

expressed in the M12.4.1.C3 cell line, a derivative of a

BALB/c mouse B-cell lymphoma with mutations impair-

ing surface expression of H2-Ad and H2-Ed b-chains. The

upper panel shows a similar degree of MHC class II

expression in the transductants. M12.4.C3 cells were

essentially negative if stained with MHC class II-specific

antibodies (OX6 for RT1.Bb, 14-4-4S for RT1.Da and

H2-Ea and OX17 for RT1.D). The weak 14-4-4S signal,

indicated by a bold line in the M12.4.1.C3 histogram,

may reflect a residual MHC class II expression on this cell

line or some unspecific binding. In any case, neither un-

transduced nor RT1.Dl-transduced cells presented SEA or

TSST-1, while both were presented by the RT1.Bl trans-

ductants with similar efficiency as by the human RAJI

B-lymphoma. A SPEC response could only be seen with

RAJI cells as presenters.

Discussion

This study compares the presentation of bacterial SAgs by

rat MHC class II molecules RT1.Bl and RT1.Dl using

transduced mouse cell lines as presenters. The generated

cell lines were found to bind RT1.Bl- and RT1.Dl-specific

mAb, respectively, and, to present peptide antigens as well

as bacterial SAgs to RT1.Bl- and RT1.Dl-restricted T-cell

hybridomas.

Binding analyses revealed the cross-reactivity of various

antibodies specific for mouse MHC class II antigens with

either RT1.Bl or RT1.Dl. H2-A-specific mAb cross-reacted

with RT1.Bl and the mAb specific for H2-E reacted with

RT1.Dl. At least in the case of the mAb 14-4-4S the cross-

reactivity between H2-E and RT1.Dl was expected, given

its known binding to rat MHC class II and its specificity

for the highly conserved H2-E-encoded Ia.7 determinant.

Some of the RT1.B-specific mAbs have been reported to

be allele-specific in the mouse; it would be of interest to

learn whether these polymorphic determinants have been

conserved between rats and mice and whether they can

discriminate rat RT1.B/D alleles as well.

Furthermore, the RT1.Bl-transduced and RT1.Dl-trans-

duced L929 lines provide a valuable tool for the determi-

nation of MHC isotype specificity in antibody binding

and MHC-restricted antigen recognition, and will be use-

ful for many researchers because RT1.Bl and RT1.Dl are

expressed by widely used rat strains such as LEW, F344

1011000

200

Cou

nts

Cou

nts

200RT1B RT1B

RT1D RT1D

0

0

200

200

0

CD74 MHC II

102 103 104 101100 102 103 104

FL1-Height FL2-Height

Figure 4. Major histocompatibility complex (MHC) class II expres-

sion before and after CD74 transduction. The left histograms indi-

cate fluorescence of the enhanced green fluorescent protein (EGFP)

reporter before (thin line) or after (bold line) transduction of the

indicated L929 transductants with a bicistronic ratCD74-EGFP con-

struct. In the right panel the same cells as in the left panel were

tested for RT1.Bl expression (OX6), respectively, RT1.Dl expression

(14-4-S) before (thin line) or after (bold line) transduction with the

rat CD74-EGFP construct.

1200

TSST1

SEA SEA

TSST1

1800

1500

1200

900

600

300

0

pg/ml pg/ml L929 L929CD74

900

600

300

0

0 0·0001

microgram/ml

neg I neg II

RT1D I

P3/2CD80 I RAJI II

RT1B I

neg I

RT1D I

P3/2CD80 I P3/2CD80 II

RT1B I RT1B II

RT1D II

neg II

RT1B II

RT1D II

microgram/ml 0·001 0·01 0·1 1

0·0001 0·001 0·01 0·1 0·0001 0·001 0·01 0·1

0·0001 0·001 0·01 0·1 1

100

200

300

400

0

100

200

300

400

500

600

Figure 5. Interleukin-2 (IL-2) release by T-cell hybridoma 3DO54.8

stimulated with staphylococcal enterotoxin A (SEA) or T-cell hybrid-

oma FRN2.7 stimulated with toxic shock syndrome toxin 1 (TSST-1)

with rat CD74-negative (left) or rat CD74-positive cells (right) as

presenters (see Fig. 4). The left panel shows data from two experi-

ments with L929 transductants, which are indicated by full lines

(experiment 1, squares indicate DR1 transductants) or dotted lines

(experiment 2, squares indicate RAJI cells). The other cells were

L929 untransduced (circle), L929 RT1.Bl-transduced (triangle), L929

RT1.Dl-transduced (inverted triangle). The right panel shows data

from two experiments with a culture period of 40 hr (full line exper-

iment 1 and dotted line experiment 2) with CD74-transduced L929

rat MHC class II transductants and the DR1 transductant P3/2

CD80 (squares). The other cells are L929CD74 untransduced (circle),

L929CD74 RT1.Bl-transduced (triangle), L929CD74 RT1.Dl-trans-

duced (inverted triangle).


H. Dlaske et al.

and WKY.45 The dose–response curves of the T-cell

hybridoma 19 to casein presented by thymocytes or by

RT1.Dl transductants were similar, while, in comparison

to thymocytes, a nearly 100-fold higher concentration of

gpMBP was needed if RT1.Bl transductants were used as

presenters. Several possibilities can be envisaged to

explain this discrepancy: (i) transductants lack costimula-

tory molecules such as CD80/86, which are expressed by

the professional antigen-presenting cells comprised in the

thymocyte preparation and which are needed to elicit a

full response of T-cell hybridoma 53/4 but not hybridoma

19; (ii) L929 cells process gpMBP and casein equally effi-

ciently but gpMBP is more efficiently processed than

casein by the professional antigen-presenting cells in the

thymocyte preparation; and (iii) the antigen-loading

machinery found in thymocyte antigen-presenting cells,

which includes invariant chain and DM molecules,

improves presentation of gpMBP by RT1.Bl, but not pre-

sentation of casein by RT1.Dl because of some intrinsic

features of RT1.Dl. Consistent with this last possibility

would be the differential effect of invariant chain trans-

duction on surface expression of RT1.Bl versus RT1.Dl.

Staphylococcal enterotoxin B and the highly homolo-

gous SECs and SPEA activated the Vb8.2-positive T-cell

hybridoma 53 poorly if presented by rat MHC class II,

whereas presentation was very efficient by DR1-trans-

duced P3/2 cells. These data are consistent with the

high concentrations of SEB and SEC1 required to

directly stimulate total rat splenocytes or lymph node

cells5,25 and our own unpublished data on a hardly

detectable LEW rat primary T-cell response to SPEA on

the one hand and the good capacity of DR1 transduc-

tants or MHC class II-positive RAJI lymphoma cells to

efficiently present SEB or SEC1 to primary rat T cells

or T-cell hybridoma,4,25 on the other. The weak but

significant capacity of untransduced L929 cells to pres-

ent SEB to the T hybridoma 53/4 cells may be the

result of a still unknown mechanism similar to the pre-

viously described MHC class II-independent activation

of CD8+ T cells by SEB presented by certain cell types

(reviewed in ref. 47).

A pronounced preference for presentation by human

MHC class II was also found for SPEC and YPM. Given

that, with exception of YPM and MAS, the tested SAgs

are derived from human isolates of S. aureus and S. pyo-

genes, it appears very likely that the species-specific differ-

ences in MHC binding reflect the adaptation to the

human host. Consistent with this notion would be the

M12.4.1C3

100 101 102

FL2-H

Isotypes/OX6/14-4-4S/OX17 Isotypes/OX17/14-4-4SIsotype/OX6

103 104 100 101 102

FL2-H103 104 100 101 102

FL2-H

SPECpg/mlpg/ml

200

250

300

150

100

50

0

pg/ml100

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60

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20

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Microgram/ml1E

-05

0·00

010·

001

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1 1

Microgram/ml1E

-05

0·00

010·

001

0·01 0·

1

Microgram/ml0·

0001

0·00

10·

01 0·1 1

SEA TSST1

RAJIRT1DRT1Bneg

500

400

300

200

100

0

103 104

600

Cou

nts

010

0C

ount

s

120

0C

ount

s

M12.4.C3.1RT1B M12.4.C3.1RT1D

Figure 6. Major histocompatibility complex (MHC) class II expression and superantigen presentation of native or rat MHC class transduced

M12.4.1.C3 cells: staining was performed with isotype-matched antibodies (filled histogram) or indicated MHC class II-specific antibodies (open

histogram). The bold open histogram of M12.4.1.C3.RT1.D respresents staining with 14-4-S monoclonal antibody (RT1.Da/H2-Aa chain) and

the thin line staining with OX17 monoclonal antibody. The lower panel shows interleukin-2 (IL-2) production by the reactive T-cell hybridoma

with indicated superantigens. Presenting cells were: untransduced M12.4.1.C3 cells; circle, RT1.Bl-transduced M12.4.C3 cells; triangle, RT1.Dl-

transduced M12.4.C3 cells; inverted triangle, RAJI; squares. Data for staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1

(TSST-1) are mean values of three experiments with standard deviation. Data for streptococca pyrogenic exotoxin C (SPEC) are from two experi-

ments distinguished by full and dotted lines.



good presentation by RT1Dl of the rat pathogen-derived

MAS, although this host adaptation may differ for the

various classes of SAgs with their highly different modes

of MHC and TCR interaction.

The complete lack of SEA and TSST-1 presentation by

RT1.Dl was striking. For TSST-1 this result was not com-

pletely unexpected, because in mouse H2-A binds TSST-1

clearly more efficiently than H2-E,21 but for SEA efficient

binding to both DR and H2-E has been reported.22,48 A

notable exception in the high-affinity binding of SEA to

DR is the DR53w molecule, which carries a histidine to

tyrosine substitution at position 81 of the b-chain, which

abolishes the Zn2+-coordinated MHC class II binding of

the SEA.49 This histidine 81 is maintained in the RT1.Dl

molecule but the two neighbouring positions are changed

(arginine to lysine at position 80 and asparagine to aspar-

tate at position 82) and may well interfere with the SEA

binding. The importance of this MHC class II region is also

highlighted by its extreme conservation because with the

exception of RT1.Dl all functional antigen-presenting

mammalian MHC class II molecules currently found in the

database (October 2008) have preserved arginine 80 and

asparagine 82. Furthermore, an arginine 80 to serine muta-

tion has been claimed to be responsible for the impaired

surface expression and intracellular accumulation of H2-Ad

and H2-Ed in the M12.4.C3 mutant.39 It will be interesting

to investigate how these unique substitutions relate to our

findings on the impaired SEA presentation and the weak

effect of CD74 on RT1.Dl surface expression.

Acknowledgements

T.H. would like to thank Niklas Beyersdorf for helpful

comments on the manuscript.

Disclosures

Supported in part by Deutsche Forschungsgemeinsachft,

SFB 571.

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