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.
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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.
e574 � 2009 Blackwell Publishing Ltd, Immunology, 128, e572–e581
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
50
40 Cou
nts
Cou
nts
30
20
10
0
Cou
nts
30
20
10
0
80
70
60
50
40
Cou
nts
30
20
10
0
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.
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Superantigen-presentation by rat MHC class II
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).
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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)
100
80
60
40
20
120
100
80
60
40
20
5
10
15 ControlRT1BRT1DHLA-DR1
20
30
40
50
60
10
120
100
80
60
40
20
120
100
80
60
40
20
120
100
80
60
40
20
120
100
80
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.
� 2009 Blackwell Publishing Ltd, Immunology, 128, e572–e581 e577
Superantigen-presentation by rat MHC class II
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).
e578 � 2009 Blackwell Publishing Ltd, Immunology, 128, e572–e581
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
80
60
40
20
0
Microgram/ml1E
-05
0·00
010·
001
0·01 0·
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.
� 2009 Blackwell Publishing Ltd, Immunology, 128, e572–e581 e579
Superantigen-presentation by rat MHC class II
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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Superantigen-presentation by rat MHC class II
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