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STUDY OF TCR V6 USAGE IN SUPERANTIGEN-REACTIVE
HUMAN T CELLS BY THE RT-PCR METHOD
Hidehito KATO, Wakae FUJIMAKI, Hisashi NARIMATSU*, Junji YAGI,
Ken'ichi IMANISHI and Takehiko UCHIYAMA
Department of Microbiology and Immunology, Tokyo Women's Medical College
*Institute of Life Science, Soka University
(Received June 22, 1994)
Human T cells reactive with several bacterial superantigens were examined in terms of the T cell receptor for immunogen (TCR) Vrs repertoires. Human eral blood mononuclear cells were stimulated with 10 ng of staphyloeoccal otoxins A, B, C2 and E (SEA, SEB, SEC2 and SEE) or toxic shock syndrome 1 (TSST-1) per ml for 3 days. The Iarge lymphoblasts recovered were expanded for 2 days in the presence of 100 U of recombinant human IL-2 per ml. T cell blasts obtained were examined for TCR V6 usage by the reverse transcriptase polymerase chain reaction method.
T cells reactive with SEA were V61', V65.2=3', VP6.1-3', V67', V69' and V618'. Those reactive with SEB were Vrs3', V612', V613.2', V614', VP15', VP17' and VIB20'. Those reactive with SEC2 were V67', VB9', VP12", VP13.2',
VP14', VP15', V617' and V620'. Those reactive with SEE were V65.1',
V66. 1-3', Vfi8' and V618', and those reactive with TSST-1 were V62' and Vrs4" .
While the present study supported principally the validity of the previous studies by
other researchers, it revealed the additional TCR V6 usage in human T cells reactive with several bacterial superantigens. I.t seems likely that T cells from Japanese in the present study and T cells from Caucasians may respond differently
to bacterial superantigens.
Introduction
A number of bacterial exotoxins have been
classified as superantigeni)'-3). These exotoxins
contain toxic shock ,syndrome toxin-1 (TSST-1), staphylococcal enterotoxins ･A-E (SEA,
SEB, SEC, SED and SEE), streptococcal
pyrogenic exotoxins A-C (SPE-A, SPE-B and
SPE-C), and a recently found Y2rsinia
Pseuclotuberczalosis-derived mitogen (YPM)`)-6).These exotoxins bind directly to major his-tocompatibility complex (MHC) class II
mole-cules7)-i3> and activate a vast number of T cell
clones in a T cell receptor for immunogen
(TCR) Vfi-selective way5)6)i`)-20) in association
with MHC class II molecules on accessory cells
(AC)8>9>. The potent T cell-stimulating activity
of these exotoxins has been implicated as the pathogenic factor in exotoxin-induced
illnes-ses')2i)22) such as toxic shock syndromei scarlet
fever and M Pseuclotubercufosis infection. The
last of these has often been reported in Japan.
Almost all of the TCR V6 repertoires in murine and human T cells reactive with these superantigenic exotoxins have been determined during the 6 years since the proposal of the concept of superantigeni`). In our preliminary experiments using the revetse
transcriptase-polymerase'chain reaction (RT-PCR) method, however, we found selective elevation of a certain Vfi element in SEA-reactive human T
cells, whic'h was,not reported previously. Th･is
observation suggests the necessity of reexami-nation of the previ6us reports (summarized in
reviewsi)-3)). ,,In thg. .present study, we examined the TCR V6 usage in human.T, cells reactive with a number of bacterial superantigens,
TSST-1, SEA, SEB, SEC and SEE. ･'
Materials and Methods
Superantigens and other reagents
TSST-1 was purified from the culture fluid of SlaPltylococcus azareus FRI169 as reported
previ-ously23). SEA, SEB, SEC2 and SEE were pur-chased from Toxin Technology (Sarasota, FL). The RPMI 1640 culture medium uSed contained 100 ptg of streptomycin per ml, 100 units of
penicillin per'mi, 10% fetal calf serum and 5 ×
10-5 M 2-ME. Recombinant human interleukin 2 (rlL-2) was kindly provided by Takeda
Chemi-cal Industries Ltd. (Osaka, Japan).
tt
Lymphoid cells' '
Peripheral blood mononublear cells (PBMC) were obtained from peripheral blood of healthy donors by Ficoll-Conray gradient seParation as
reported previously2`)25). PBMC (1･xy2 × 106/ml)
were stirbulated with various doses of
super-antigens or 20 ng 'of CD3 monoclonai
anti-body (mAb) OKT3 per ml for 3 days. Recovered
cells were subjected to, Percoll density gradient
centrifugation (Percoll density of 1.068). The large lymphoblasts fractionated were expanded
for 2 da･ys in the presence of 100 U of rlL-2 per ml. The cells recove'r'ed were ftacti6nafed int6 large lymphoblasts. The･ large lymphoblasts
obtained'contained 60-v90% CD3' cells as
determined by flowcytometric analysis using anti-CD3` mAb.
Assay for IL-2 production ･
･PBMC were stimula･ted with varying
concen-trationS'of the,.bacterial superantigens in 1-ml
quantities' in 24-well Falcon plates (Becton Dickinson, San Jose, CA) for 24 hr. ･Culture
supernatants were collected and assayed for
2 activity by using IL-2-dependent CTLL-2 as reported previously2`). Data are presented as
units/ml.
RT-PCR method for. determination of TCR
/tt
V6 usage
TCR V6 usage in superantigen-reactive
human T cells was determined by the RT-PCR methbd originally described by Choi et al.'6) with slight modifications. Total mRNA was prepared from superantigen- or anti-CD3
induced T lymphoblasts by using oligo
de6xythymidine-conjugated magnetic beads
(Dynabeads Oligo (dt) 25: Dynal, Oslo, Norway).
cDNAs were synthesized by incubating the
total mRNA obtained from each T lymphoblast sample with 10 units of reverse trapscriptase (RAV2: Takara, Kyoto, Japan) in the final volume of 25 pt1/tube f6r 90 min at 420C. Then aliquots of each cDNA sample (final volume, 20 rd/tube) were amplified with various numbers of incubation cycle by using 22 5'V6-specific
sense primers and the 3'Crs-specific anti-sense .priiner in the presence o'f 1 unit of AmpliTaq
DNA Polymerase (Perkin-Elmer Cetus mertts, Norwakl, CT) in Program Temperature
Control System 'PC-700 (ASTEC, Fukuoka,
Japan). TCR Ca cDNA as an internal control was co-amplified in each reaction mixture by using the 5'Ca sense pr'imer and the 3'Ca antisense primer.'For quantitation of the
plified products, the 3'primers end-labeled with
32P were used. The sequences of the specific primers used were describ6d by Choi et al.i6), and are shown in Table 1. All of these primers
.woul.d be expected to cg"vugr at leqst 80% of the
human TCR V6 gene segments. The amplif･ied products were electrophoresed in 2.5% agarose
gels, dried and exposed on iMaging plates <Fuji
Photo Film Co., Tokyo, Japan). The Vrs and Ccr bands identified were examined for ity expressed as counts per minute (cpm) of photostimulated luminescence by a Bioimaging Analyzer-, BAS 2000 (Fuji Photo Film Co.). For
each sample, the radioactivity in''the V6 : Cev ratio (=1000 × radioactivity in V6
band/radio-Table 1 Sequences of primers used for the
RT-PCR
Primer Vfi1 VP2 Vfi3 VP4 VP5.1 VP5.2 VP6.1-3 VS7 V68 V69 V610 V611 VfiI2 Vfi13.1 Vfi13.2 VP14 VP15 V616 V617 VP18 Vfi19 Vfi20 3,CP 5'Ca 3'Ca 5'-3'sequenceGCACAACAGTTCCCTGACTTGCAC
TCATCAACCATGCAAGCCTGACCT
GTCTCTAGAGAGAAGAAGGAGCGC
ACATATGAGAGTGGATTTGTCATT
ATACTTCAGTGAGACACAGAGAAAC
TTCCCTAACTATAGCTCTGAGCTG
AGGCCTGAGGGATCCGTCTC
CCTGAATGCCCCAACAGCTCTC
ATTTACTTTAACAACAACGTTCCG
CCTAAATCTCCAGACAAAGCTCAC
CTCCAAAAACTCATCCTGTACCTT
TCAACAGTCTCCAGAATAAGGACG
AAAGGAGAAGTCTCAGAT
CAAGGAGAAGTCCCCAAT
GGTGAGGGTACAACTGCC
GTCTCTCGAAAAGAGAAGAGGAAT
AGTGTCTCTCGACAGGCACAGGCT
AAAGAGTCTAAACAGGATGAGTCC
CAGATAGTAAATGACTTTCAG
GATGAGTCAGGAATGCCAAAGGAA
CAATGCCCCAAGAACGCACCCTGC
AGCTCTGAGGTGCCCCAGAATCTC
TTCTGATGGCTCAAACAC
GAACCCTGACCCTGCCGTGTACC
ATCATAAATTCGGGTAGGATCC
120 1OO=
E) 80
v
5 'g 608
9 4o Q ny - 20 oO O.Ol O.1 1 10 100
Concentration of exotoxins (nglml)Fig. 1 Indllction of IL-2 production in human PBMC by various superantigens
Human PBMC (2 × 1061ml) were stimulated
with SEA (A), SEB (Q), SEC, ('[]) or TSST-1 (e)
at the indicated doses for 24 hr and culture
supernatants were examined for IL-2 activity.
Quoted from the study reported by Choi et al.i6)
activity in Ca band). The
t-test was used for statistical
two-tailed paired
anaiysis.
Results and Discussion
T cell-stimulatory activity of various bacterial
superantigens
Initially SEA, SEB, SEC2 and TSST-1 were examined for their ability to induce IL-2
pro-duction from T cells of PBMC. Human PBMC
were stirriulated in vitro with various
concen-trations of the superantigens for 24 hr and examined for IL-2 production. They induced the
production of substantial amounts of IL-2 at 100
pg/ml or more (Fig. 1), and these four toxins and SEE induced blast formation in very high levels at 10 ng/ml on day 3 after stimulation (data not shown): The results ･show that these
five bacterial superantigens are potent T cell activators at similar levels. In the following experiments for determining TCR VP
reper-toires used in the superntigen-reactive T cells,
PBMC (2 × 106/ml) were stirnulated with 10 ng
of superantigen per ml to obtain T
lymphoblast-enriched cell preparations.
Determination of the optimal number of incu-bation cycles for performing RT-PCR
analy-sis
Total mRNA was prepared from samples of SEB-induced T lymphoblasts and then single-stranded cDNA was synthesized. We chose one T cell sample with a high RNA content and another with a low RNA content. An aliqubt of each synthesized sample of cDNA in the pre-sence of reverse transcriptase was amplified with various numbers of incubation cycles by
using the 5'Cev sense primer and 3'Ca antisense
primer, using the Program Temperature
Con-troller, and examined for the level of amplified
TCR Ca and TCR Vfi 3 genes. The results
showed that the Cev gene and the VB 3 gene were amplified in a proportional relationship with each other between 23 and 26 incubation cycles in both samples (Fig. 2). The results
indicate that the RT-PCR method can be
applied to samples containing various amounts of cDNA. In the following experiments, the
1OOOO
A
Ea
ov
oo = m U co pt .EE. iooo
8
:. e tlpe
sog
1OO23 24 25 '26 23 24 25
lncubation cycles lncubation cycles
Fig. 2 Determination of optimal number of incubation cycles for performing the
RT-PCR
Human PBMC (2 × 106/ml) were stimulated with 10 ng of SEB per ml for 3 days. Total mRNA was prepared from two samples of SEB-induced T blasts, and Ca (!]) and VP3 (-) were ampljfied wjth varlous numbers of
incubation cycles. T celtsarnple A has a high RNA content and sarnple B had
a low RNA content.
26
amplification was done with 25 cycles without consideration of the RNA content of the
sam-ples.
Identification of TCR V6 elements used by human T cell populations reactive with SEA,
SEB, SEC,, SEE and TSST-1
T cell lymphoblasts induced by 10 ng of SEA,
SEB, SEC2, SEE or TSST-1 per ml were
examined for their TCR VB usage by the
RT-PCR method. T cell blasts induced by anti-CD3 mAb were used as a control. The V6 : Ca ratios calculated from autoradiograms of am-plified TCR transcripts of T Iymphoblasts in-'duced by these- superantigens from a single
donor are presented in Table 2. When the
ratios in both the control･ and experimental samples were less than 50, we removed these data from consjderatjon as values too Iow to
evaluate. The ratios of VPI : Ca, V65.2-3 : Ca,
V66.1-3:Ca, VP7:Cev, V69:Cev and VP18:
Cev were higher in the SEA-induced T cell blasts than in the control CD3-induced T cell
blasts. This increase in the V618 : Cev ratio was reported previously'9). V6 : Ca ratios in other
V6s in the experimental samples were far
below those of the control. Ih SEB-induced T cell blasts, an increase in the ratios V6:Ca,
VP12:Ca, V614:Ca, Vfi17:Ca and V620:Ca
was observed. The level of the VB15:Ca and
VP20:Ca was observed. The level of the
V613.2:Ca ratio in the experimental sample was the same as that in the control. We con-sider the results as evidence that V613.2' T
cells are reactive with SEB in that the ratio in
the former was not lower than that in the
control. The reactivity of VB13.2' T cells with
SEB was not reported previouslyi6). In SEC2-reactive T lymphoblasts, the ratios VP9 : Cev,
V612:Ca, VP13.2:Ccr, VB15:Ca, VP17:Ccr
and VP20:Ccr were increased. The Vfi7:Ca
ratio was high in the control･sample and in-creased slightlyin the experimental sample. We
consider these findings as evidence that V67' T cells react with SEC2. The increase in the ratios
V67 : Ccr and V69 : Ca was not reported previ-ouslyi6). In SEE-induced T lymphoblasts, the
ratios V65.1:Ca, V66.1-3:Ca, V68:Ca and
Table2 TCR V6 usage in human T cells
reactive with SEA, SEB, SEC,, SEE and TSST-1
Vp:Ca ratio
Vfielement
Tcell stimulants
anti-CD3 SEA SEB SEC, SEE TSST-1
1 61 100 28 30 6 25 2 475 35 88 71 55 2276, 3 ,.665 33 922 139 89 107 th 4 96 17 23 2 27 (12e)-5.1 158 43 31 9 258th 43 5.2-3 139 311 49 93 77 50 6.1-3 414 631nt 130 320 807- 86 7 416 510 83 (430) 392 35
-
-8 60 32 40 24 264 22 -9 59 169 29 (82) 52 38 un-10 12 37 32 31 22 19 11 7 26 30 11 24 32 12, 12 18 78 127 19 15
m
-13.1 150 16 34 64 20 28 13.2 62 11 (62)- 102 23 11 14 91 33 183 205 15 34 rm 15 60 29 128 146 5 8-16 34 36 21 20 18 18 17 34 2 58 139 19 24 -18 90 (198) 27 36 256 34
m
19 30 9 44 20 21 26 20 112 1 154 238 5 18-Human PBMC from a healthy donor (2×1061ml) were
stimulated with 10ng of SEA, SEB, SEC2, SEE or TSST -1 per ml for 3 days. The recovered cells (2×.105/ml) were expanded for 2 days in the presence of 100U of rlL-2 per ml, The T lymphoblast-enriched preparations
obtained were tested for TCR VP usage. Data are
expressed as VP:Ca ratios (cpm VP/cpm CaX
1,OOO). For the underlined ratios, we consider that reactivity with the correspQnding superantigen was
present. The parentheses indicate that the
superantigen-reactivity was not reported previously.
In TSST-1-induced T lymphoblasts, the ratio$
VP2 : Ca and V64 : Cev were also higher than in the-controls. The increase the V64 : Ccr ratios was also not reported previously'6).
The V6 usage of T cell blasts induced by these superantigens was examined in T cells from different donors and the data are summar-ized in Fig. 3. The data are expressed as the VP : Cev ratio. With regard to the VP elements which were reported in the previous studies to be involved in the reactivity with these five superantigens, our data for the several donors
are compatible with the results of other studies
with one exception. The V67:Ca ratio in
SEA-reactive T cells differed between two donors: an increase in one donor and a slight decrease in an other donor; the ratio in the control was high and ,the decrease is marginal in. the experiment samples. We consider that VP7' T cells in the second donor were reactive
with SEA. With regard to the VP elements
which we found newly in the present
experi-ments to be involved in the reactivity with the
superantigens, it seems necessary to discuss several points. For the V618' element in SEA-reactive T cells, the V618:Ccr ratio was
in-creased in both of two donors. For the Vfi13.2'
element in SEB-reactive T cells, the V6 t' Ca ratio was increased in three donors, not chan-ged in two and slightly decreased in one. We consider that the Vrs13.2' T cells of the last donor would react with SEB on the basis of the previous discussion. With regard to VP7' and VB9' elements in T cells reactive with SEC2, we examined only one donor. Repeated experi-･ments seem to be necessary to conclude that
Vfi7' and V69' T cells are reactive with SEC2. For the Vfi4' element in T cells reactive with TSST-1, the V64:Ccr ratio was increased in three donors, not changed in two and slightly decreased in one. We consider that V64' T cells of the Iast donor would be reactive with
TSST-1 on the basis of the previous discussion.
Effect of concentration of SEA on the reper-toire in the SEA-reactive T cells
We thought it was important to know
whether or not the V6 repertoire in the
superantigen-reactive T cells is influenced by the concentration of the superantigens.
Pro-vided that preferential activation is observed in
T cells bearing particular TCR VP elements at
a lower toxin dose, what is meant by the results would be that binding affi-nity for the complex
of superantigen/MHC class II molecules differs among TCR V6 elements responsible for the
recognition of the superantigen.
Human PBMC were stimulated with varying
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頃Table 3 TCR Vfi usage in T lymphoblasts induced by various concentration of SEA
RatiosofVfifCa vfielement SEA(ng/ml) anti-CD3 20ng/ml O.1 1 10 leo 15.2-3 6.1-3 7918 113 152 325 450 79 71 l13 439 839 793 243 296 90 226 479 691 218 336 153 362 818 706 240 287 195 265 504 684 292 308
Human PBMC (2×106/ml) from a healthy donor were
stimulated with the indicated doses of SEA for 3 days
and T lymphoblasts were prepared according to
Mate-･rials and Methods and tested for TCR Vrs usage, Data are expressed as V6 : Ca ratios.
and the T lymphoblasts obtained were
examined for V6 usage (Table 3). The results show that the V6 repertoire observed at a high SEA concentrations. The results sugge$t that the binding affinity for the SEA/HLA class II
complex does not differ among TCR V61,
VP5.2-3, Vfi6.1-3, V67, V69 and V618. Several explanations may be possible for the
partial discrepancies in the TCR Vfi repertoires
used in human T cells reactive with SEA, SEB, SEC2 and TSST-1 between the present study
and past studies by other researchers. It seems
unlikely that the technical difference between
the present study and studies by other
researchers caused the discrepansies. The cul-ture method for preparing the toxin-reactive T lymphoblasts and the method of RT-PCR
analy-sis used in the present study were identical to
those by other researchers. As a small
differ-ence cDNAs were synthesized from total
mRNA in the present study and from the total RNA instead of total mRNA in the studies of other researchers. Primers used in the present
study were same as those used by other
researchers. The most plausible explanation may be as follows. The ratios Vrs13.2:Ca in
SEB-induced T lymphoblasts, VP7:Ccr and
V69 : Ca in SEC2-induced T lymphoblasts and
V64:Ca in TSST-1-induced T lymphoblasts
were not elevated in several cases in the present
study. These variable results may have Ied to the different conclusions between the present and previous studies. Second, the V6-selective response to superantigens may be influenced in some degree by the difference in human races.
T cells from Japanse in the present study and T
cells from Caucasians may respond differently
to bacterial superantigens.
Acknowledgements
This work was suppoted by grants from Ministry of
Education, Science and Culture of Japan, and Ministry of Health and Welfare of Japan
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RT−PCR法を用いた，細菌性スーパー抗原反応性T細胞の 丁細胞受容体Vβエレメントの使用頻度の解析 1）東京女子医科大学 微生物学免疫学教室 2）創価大学 生命科学研究所 カトウ ヒデヒト フジマキ ナリマツ ヒサシ 加藤 秀人1）・藤巻わかえ1）・成松 久2） ヤ ギ ジユンジ イマニシ ケンイチ ウチヤマ タケヒコ 八木 淳二1）・今西 健一）・内山 竹彦1） 我々は，種々の細菌性スーパー抗原staphylococcal enterotoxin A， B， C、， E（SEA， SEB， SEC2， SEE）およびtoxic shock syndrome toxin−1（TSST−1）に反応性のヒトT細胞に発現する， T 細胞受容体（TCR）Vβエレメントの使用頻度を調べた．健常人より得られた末梢リンパ球を10ng のSEA， SEB， SEC、， SEEおよびTSST−1で3日間培養し，得られたTリンパ芽球を更に2日
