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Chain Junctional Region in Prenyl Pyrophosphate Antigen Recognition by 
T Cells1
Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells recognize prenyl pyrophosphate Ags and their
analogues in a V2V2 TCR-dependent manner. Few data are available
regarding the TCR structural requirements for recognition of such
prenyl pyrophosphate Ags by T cells. Presently, we made chain
pair switch, chimeric, and site mutant TCRs and transfected them
into TCR- mutant Jurkat T cells to examine the
effects of changing the TCR junctional region sequences on
reactivity to prenyl pyrophosphate Ags. Substitution of the TCR
junctional region (N and J) sequences from an Ag-reactive TCR with
TCR junctional region sequences from an Ag-nonreactive TCR abrogated
reactivity to the prenyl pyrophosphate Ag isopentenyl pyrophosphate and
to its synthetic analogue ethyl pyrophosphate but not to a
mycobacterial supernatant containing a mixture of prenyl pyrophosphate
Ags. Substitution of only the TCR N nucleotide region with that from
this Ag-nonreactive TCR destroyed reactivity to isopentenyl
pyrophosphate and to the mycobacterial supernatant. Substitution of the
entire V2 chain from the Ag-reactive TCR with a V1 chain from an
Ag-nonreactive TCR yielded a prenyl pyrophosphate Ag-nonreactive TCR.
Thus, using TCR mutagenesis and TCR transfectants, we show that
TCR reactivity to prenyl pyrophosphate Ags is dependent upon the
junctional region of the TCR chain and upon pairing of V2 and
V2 TCR chains. These structural requirements of TCR
recognition of prenyl pyrophosphates distinguish this reactivity from
that of protein superantigens and emphasize the importance of the
TCR CDR3 loop and adjacent residues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 46 Vß TCR gene segments
that can pair to form several thousand receptor combinations, there are
only six V and four major V gene segments used by human T
cells (1). Among these T cell gene pairs, the V2V2 TCR
pair (V2=V9 in alternate nomenclature) (2) is expressed on 50 to
75% of human peripheral blood T cells and thus comprise 3 to
5% of circulating adult human peripheral blood CD3+
cells (3, 4). The other major population of human T cells
expresses V1 paired with several V gene segments; these V1 T
cells far outnumber V2V2 T cells in the intestine (5, 6).
The predominant V2V2 T cell population is polyclonal, as judged
by its high degree of junctional diversity (7, 8, 9, 10). The diversity
regions of TCRs are encoded by V (variable), D (diversity), and
J (joining) segments (11, 12). The CDR3 loops are encoded by the V, D,
and J segment sequences in the case of TCR and by the V and J
segment sequences in the case of TCR. Further diversity is imparted
by the use of one to three D segments in tandem, by imprecise V-D
and D-J joining, by exonuclease nibbling of the joining ends, and by
the incorporation of random nontemplate-encoded N nucleotide additions
(12). Thus, despite the limited number of V gene segments, there is
enormous potential for diversity in TCRs located at the
V(D,D,D)J joining ends, referred to as junctional diversity.
V2V2 T cell numbers in peripheral blood are increased markedly
compared with those in normal controls in a variety of infectious
diseases, such as tuberculosis (13, 14), salmonellosis (15), tularemia
(16), brucellosis (17), erlichiosis (18), leishmaniasis (19), malaria
(20), toxoplasmosis (21), HIV (early stages) (22), and EBV (23). Such
expansions can be recapitulated in vitro using extracts from the
organisms causing many of these diseases as well as other organisms,
such as herpes simplex virus type 1, Listeria
monocytogenes, Escherichia coli, Streptococcus
pyogenes, and Staphylococcus aureus (7, 8, 24, 25, 26, 27, 28, 29, 30).
The Ags responsible for expanding T cells have been most
extensively studied from mycobacteria, where they were identified as
prenyl pyrophosphates (27, 31, 32, 33). One such natural Ag is isopentenyl
pyrophosphate (IPP)3, a
246-Da molecule that has a five-carbon isoprenyl chain and a
pyrophosphate moiety (33). It can be secreted by mycobacteria and is an
essential precursor for a large number of molecules, such as
cholesterol, vitamins, dolicol phosphates, and ubiquinones, in both
eukaryotic and prokaryotic organisms. TUBag 1 and 2 are another group
of closely related antigenic molecules that have been isolated from the
cytoplasm of mycobacteria. They are pyrophosphate-containing molecules
whose complete structures have not been determined (31).
Other naturally occurring but less potent Ags include
2,3-diphosphoglycerate, glycerol-3-phosphate, ribose-1-phosphate, and
xylose-1-phosphate (34). Besides these naturally occurring Ags, there
are several alkyl and alkenyl phosphate and pyrophosphate analogues
that have been synthesized and are recognized by V2V2 T cells.
The most active of these are ethyl pyrophosphate (EPP) and
monoethylphosphate (MEP) (32, 33). Both natural and synthetic Ags
require their phosphate or pyrophosphate moieties for biologic activity
based on phosphatase treatment and an analysis of synthetic compounds
(31, 32, 35). Alkyl phosphate compounds with carbon chains containing
aromatic moieties or with more than five carbon atoms are not
recognized, nor are nonphosphate-containing derivatives of these Ags
(32, 34, 36). Overall, the prenyl pyrophosphate Ags are the most
clearly defined and extensively studied Ags recognized by human
T cells.
In contrast to the uptake and processing of Ags presented by MHC class I or class II molecules, prenyl pyrophosphate Ags do not require Ag processing, since they can be presented by cells whose surfaces have been glutaraldehyde fixed before exposure to Ags. Moreover, although recognition requires cell-to-cell contact, professional APCs and known Ag-presenting molecules are not required (37, 38).
V2V2 T cells undergoing expansion in vitro in response to
mycobacteria show extensive junctional diversity and thus are
polyclonal (8, 9, 10, 39). Yet, only T cells expressing the V2V2
chain pair respond to prenyl pyrophosphate Ags from mycobacteria. This
led to the hypothesis that recognition of prenyl pyrophosphate Ags was
V gene mediated. However, most mycobacteria-reactive V2V2 T cell
clones use the J1.2 segment. In contrast, only half the V2V2 T
cells using the J1.3 or J2.3 segments react with mycobacteria (7, 9, 39, 40), suggesting that the TCR junctional region may be
important in prenyl pyrophosphate Ag recognition.
For TCRß, a critical, although not exclusive, role exists for the
CDR3 regions in recognizing peptides in the context of MHC Ags
(41, 42, 43, 44, 45, 46, 47). The crystal structures of TCRß complexed with
peptide/MHC class I Ags show that the CDR3 regions of both the - and
ß-chains are intimately involved in contacting both the peptide and
the class I molecule (48, 49). In contrast to the clear picture of the
TCRß/peptide/MHC interaction, no such analyses have been performed
with T cells and their nonpeptide ligands. TCR gene transfer
studies have shown that T cell-mediated recognition of prenyl
pyrophosphate Ags (50) and of staphylococcal superantigen A (51) is TCR
dependent, but there have been no reports of gene transfer using mutant
and chimeric TCR genes to explore the role of TCR gene
segments in the recognition of prenyl pyrophosphate Ags.
Using TCR gene transfer studies, here we show that reactivity to prenyl
pyrophosphate Ags requires expression of the V2 chain and
coexpression of V2. Substitution of either chain, TCR N
nucleotide-encoded sequences or of an entire TCR junctional region
for another, changes the reactivity to prenyl pyrophosphate Ags. These
data show that the TCR junctional region plays a crucial role in the
TCR-mediated recognition of phosphate Ags, as is the case for
conventional recognition of peptide-MHC complexes by TCRß.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Jurkat J.RT3-T3.5, obtained from American Type Culture Collection (Rockville, MD), or Jurkat 31.13 (a gift from Dr. Ellis Reinherz, Dana-Farber Cancer Institute, Boston, MA) are TCRß-negative variants of Jurkat that lack cell surface TCR expression. These cells and transfectants derived from them were maintained on RPMI 1640 supplemented with 10% bovine calf serum, 10 mM HEPES, penicillin-streptomycin, 5 x 10-5 M ß-ME, and L-glutamine. EBV-transformed lymphoblastoid cell lines were derived as previously described and were maintained on RPMI. SH-5YSY neuroblastoma cells were obtained from Dr. Gloria Lee, Harvard Medical School (Boston, MA), and were maintained on RPMI. The Va2 human fibroblast cell line was obtained from Dr. Charles Stiles (Dana-Farber Cancer Institute) and were maintained in DMEM with the additives listed above.
Ab and Ag reagents
mAb ascites against T cell Ags used were as follows: control mAb
(P3), pan TCR (anti-TCR1), V1/V1 (TCS1), V2
(BB3), V2 (7A5), and CD3 (OKT3). The specificity of these mAbs was
previously reviewed (1). FITC-conjugated F(ab')2 goat
anti-mouse IgG was purchased from Tago (Burlingame, CA). The
mycobacterial supernatant was obtained by culturing Mycobacterium
fortuitum in Middlebrook 7H9 broth (Difco, Detroit, MI) with 0.5
g/l Tween-80 and 2 ml/l glycerol under agitation at 37°C until
stationary phase growth was observed. Two weeks later, mycobacteria
were removed by centrifugation, followed by 0.2-mm pore size
filtration. This mycobacterial supernatant was then purified as
previously described (33). This preparation had primarily a 276-Da
molecule closely related to IPP (33). EPP was synthesized as described
previously (32). IPP was purchased from Sigma (St. Louis, MO). In some
experiments, Ag preparations were either mock treated or treated with 8
U of shrimp alkaline phosphatase (Sigma) in a volume of 500 µl for
2 h at 37°C.
Derivation of TCR cDNA
cDNA synthesis was performed as previously described (50).
Briefly, mRNA was extracted from 4 x 107 T cells
by the acid guanidinium isothiocyanate method. First-strand cDNA was
synthesized from 10 µg of total RNA using AMV reverse transcriptase
(Promega, Madison, WI). This cDNA was used as template to obtain
full-length TCR - and -chains by PCR, using oligonucleotide
primers within the 5' untranslated regions of the appropriate V and
V gene segments and 3' untranslated regions of the C and C
gene segments. The TCR gene PCR product was subcloned as an
XhoI-BamHI fragment into the expression plasmid,
pCDLSR296, downstream from the SV40/HTLVI hybrid promoter. The
gene PCR product was subcloned as an XhoI-XbaI
fragment into the plasmid pSRneo. PSRneo was derived by replacing
the spleen focus forming virus long terminal repeat promoter of the
mammalian expression vector pFneo with the SV40/HTLVI hybrid promoter
from pCDLSR296 (H. Band, unpublished observation).
Derivation of the V2/W/J1.2 transfectant
This transfectant has been described previously as DBS43 and was
made by cotransfecting J.RT3-T3.5 cells with TCR and TCR cDNAs
from the V2V2 T cell clone DG.SF13 (50).
Derivation of the V2/GN/J1.3 transfectant
The TCR sequence from the T cell clone PBLC1 has a sequence
that is identical with the TCR sequence of the T cell clone DG.SF13,
except for the N nucleotide region and the J region (52). This cDNA
was cotransfected into Jurkat 31.13 with the cDNA encoding the TCR
chain from DG.SF13 to yield the transfectant V2/GN/J1.3.
Derivation of the V2/GN/J1.2 transfectant
The TCR chain was made using a two-step PCR. In the first
step, cDNA from wild-type PBLC1 TCR, which differs from DG.SF13
only in the N and J regions, was used as a template, with a 5' primer
(5'-GGGCTCGAGGACACCGCTTTACAACGA-3'; primer 1) occurring within the
5' untranslated region of V2 and a 3' primer
(5'-GCCCAACTCATTTCCCTCCAACAACGGACA-3') to
generate: V2 N J1.2.........CALWE
GN ELG
A separate PCR was employed to generate cDNA encoding the remainder of
the DG.SF13 wild-type -chain. Full-length DG.SF13 TCR cDNA was
used as a template. The 5' primer
(5'-GAGTTGGGCAAAAAAATCAAGGTATTTGGT-3') and a 3' primer occurring in
the untranslated region of C (5'-GGGTCTAGAGTGAGGTTCTCTGTGT-3';
primer 2) were used to
generate: J1.2ELGKKIKVFG.......
The above complementary PCR products were annealed and used as a
template in a second step PCR using primers 1 and 2 to generate a
full-length chimeric -chain (V2/GN/J1.2) differing from the
wild-type DG.SF13 only in the N region (substitutions are
underlined): V1.2 N
J1.2....CALWE GN ELGKKIKVFGP.....
This chimeric cDNA was cotransfected with DG.SF13 TCR cDNA into
Jurkat J.RT3-T3.5 cells to give V2/GN/J1.2.
Derivation of the V2/W/J1.2/V1 transfectant
TCR cDNA from DG.SF13 was cotransfected with TCR cDNA from
the prenyl pyrophosphate Ag-nonreactive V1V1 T cell clone named
SP-F7 (50) into 31.13 cells.
Transfections
Stable transfectants of the ß- Jurkat
mutants J.RT3-T3.5 or 31.13 were derived by electroporation of
107 cells with 25 µg of pFneo having TCR inserts
together with 100 µg of pSR having TCR inserts (50, 53). The
cells were plated into two 24-well plates and selected in 1 mg/ml G418
(Life Technologies, Grand Island, NY) beginning on day 2. Wells
containing TCR transfectants were identified by flow cytometry
with the pan TCR mAb, anti-TCR1. TCR transfectants
were positively selected using magnetic beads after treatment with the
anti-TCR1 mAb and then were cloned by limiting dilution using
irradiated (5000 rad) Va2 human fibroblast feeder cells. Cloned
transfectant cells were tested for signaling through the TCR by
determining their IL-2 release in response to stimulation with
anti-TCR1 mAb plus 10 ng/ml of PMA, as described below. Only
transfectants with similar IL-2 release responses to anti-TCR1
were compared with each other for reactivity to prenyl
pyrophosphate Ags.
Stimulation of Jurkat transfectants
Stimulation of T cell clones and Jurkat transfectants was performed in 96-well flat-bottom plates with 1 x 105 responder cells/well in 0.2 ml (50). In some experiments, 5 x 104 mitomycin-treated or glutaraldehyde-fixed B lymphoblastoid cells or SH-5YSY neuroblastoma cells per well were used as feeders or APC, but these APC were not necessary to obtain IL-2 release from the transfected Jurkat cells. Half-log dilutions of Ag or, as a positive control, the calcium ionophore, ionomycin (at 1 mg/ml), were added in the presence of 10 ng/ml PMA as a costimulator (54). After 24 h, supernatants were harvested and were tested at a final dilution of 1/8 for their ability to stimulate the growth of the IL-2-dependent HT-2 cell line. Proliferation assays were performed in triplicate using 5 x 103 HT-2 cells/flat-bottom well of a 96-well plate. After 18 h, the cells were pulsed with [3H]thymidine (1 µCi/well), harvested at 24 h, and counted by liquid scintillation. The variability in the absolute counts per minute data we occasionally observed between experiments resulted from day-to-day variability in the cell culture densities of the Jurkat transfectants and in the HT-2 indicator cell line used in the biologic assay for IL-2. Despite this variability in the absolute counts per minute, the differences in Ag reactivity between the transfectants were consistent in all experiments. Error bars indicate the SEM of triplicate samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain junctional region from a prenyl
pyrophosphate Ag-reactive TCR with that from a prenyl pyrophosphate
Ag-nonreactive TCR alters the pattern of reactivity to prenyl
pyrophosphate Ags and their analogues
To name the TCR transfectants used in this study, we use a
descriptive designation that shows the V/N/J segment use of the TCR
chain. Thus, transfectant V2/W/J1.2 uses the V2 segment, with
a tryptophan (W, single letter amino acid code) in the N region, and
rearranges to the J1.2 segment (Tables I and
II). Unless
otherwise noted, the TCR chain expressed in the transfectants is the
wild-type V2 TCR chain from prenyl pyrophosphate-reactive TCR clone
DG.SF13 (Table I
) (50). Previous studies have shown that cotransfection
of the V2 and V2 TCR cDNAs from the mycobacteria-reactive clone
DG.SF13 confers phosphate Ag reactivity upon a TCR-
mutant of Jurkat T cells. This transfectant, V2/W/J1.2
(originally designated DBS43) (50), releases IL-2 in response to prenyl
pyrophosphate Ags including those found in mycobacterial extracts, IPP,
and to the synthetic alkyl phosphates, MEP and EPP. To examine the
possible role of the TCR chain junctional region in the recognition
of phosphate Ags, we made a transfectant designated V2/GN/J1.3
with the same TCR chain as the prenyl pyrophosphate-reactive
transfectant V2/W/J1.2, but with a TCR chain that differs from
that of V2/W/J1.2 only in the junctional region. The native T
cell clone, PBLC1, expresses this TCR chain paired with a TCR
chain using a V1 gene segment (52). This TCR chain from
V2/GN/J1.3 has the same variable and constant region sequence as
that of V2/W/J1.2, but uses J1.3 instead of J1.2, and the N
nucleotide region is GN instead of W (Tables I and II).
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2/W/J1.2 and the V2/GN/J1.3 transfectants paired with
the same V2 chain were compared in IL-2 release assays for their
ability to respond to the synthetic prenyl pyrophosphate Ag analogue,
EPP (see Fig. 1 for structure of prenyl
pyrophosphate Ags and their analogues used in this study). Although
both V2/W/J1.2 and V2/GN/J1.3 transfectants responded
similarly to anti-TCR mAb over a 2-log10 range of mAb
dilutions, only V2/W/J1.2 released IL-2 in response to
EPP, with a half-maximal response seen at approximately 35 µM (Fig. 2). These results show that the specific
sequence of N- and J-encoded residues is necessary for reactivity
to EPP.
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2/GN/J1.3 transfectant was capable of
recognizing naturally occurring prenyl pyrophosphate Ags, we tested its
reactivity to a partially purified supernatant from a culture of
M. fortuitum that is devoid of protein and that contains IPP
and a closely related 276-Da molecule (tentatively identified as an
aldehyde derivative of IPP) (33) (C. T. Morita, unpublished
observations). The V2/W/J1.2 transfectant released IL-2 in
response to EPP and to the mycobacterial supernatant over a
1-log10 range of Ag dilutions (Fig. 3, upper panel). In
contrast, the V2/GN/J1.3 transfectant made no detectable IL-2 in
response to EPP, while retaining responsiveness to the mycobacterial
supernatant over the same 1-log10 range of Ag dilutions as
the V2/W/J1.2 transfectant (Fig. 3, lower
panel). Thus, the wild-type V2/W/J1.2 and the
chimeric V2/GN/J1.3 transfectants both responded to the
mycobacterial supernatant in a dose-dependent fashion, although the
latter had higher baseline IL-2 release. Since the mycobacterial
supernatant contains IPP, we tested V2/GN/J1.3 for reactivity to
this Ag in purified form. Whereas the V2/W/J1.2 transfectant
reacted to both EPP and IPP over a 2.5-log10 range of Ag
dilutions (Fig. 4, upper
panel), V2/GN/J1.3 failed to release detectable
levels of IL-2 in response to either EPP or IPP (Fig. 4, lower panel). Thus, the V2/GN/J1.3 TCR must
recognize another constituent of the mycobacterial supernatant, perhaps
the 276-Da molecule (Fig. 3, lower panel). In support
of this possibility, alkaline phosphatase treatment of the
mycobacterial supernatant totally abolished its ability to stimulate
either the V2/GN/J1.3 or the V2/W/J1.2 transfectants (Fig. 5). These experiments show that a change
in the TCR junctional region sequence alters the pattern of
reactivity to prenyl pyrophosphate Ags and their analogues.
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chain N nucleotide region from a
prenyl pyrophosphate Ag-reactive TCR with that from a prenyl
pyrophosphate-nonreactive TCR abrogates reactivity to prenyl
pyrophosphate Ags and their analogues
To examine the relative roles of the N nucleotide- and
J-encoded regions in reactivity to these prenyl pyrophosphate Ags,
we generated a chimeric TCR chain composed of the J1.2 region
from the Ag-reactive V2/W/J1.2 and the N nucleotide region from
the Ag-nonreactive V2/GN/J1.3. This chimeric TCR chain was
cotransfected into TCR- recipients with the V2
chain from the phosphate Ag-reactive V2/W/J1.2 (Tables I and II).
The resultant transfectant, termed V2/GN/J1.2, had the same TCR
sequence as the Ag-reactive V2/W/J1.2, except for a substitution
of GN for W in the N nucleotide region of the TCR chain. This
chimeric transfectant responded to anti-TCR mAb over a
2.5-log10 range of mAb dilutions as did the wild-type
V2/W/J1.2, but failed to make detectable IL-2 in response to EPP
(Fig. 6, lower panel),
IPP, or the mycobacterial supernatant (data not shown). Thus, a
two-amino acid substitution in the area within the TCR CDR3 loop
encoded by the N nucleotide region abrogated reactivity to prenyl
pyrophosphate Ags and their analogues.
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chain is necessary for reactivity to prenyl pyrophosphate
Ags
For viral and bacterial superantigen reactivity, the TCRß or
TCR chain confers reactivity, since pairing of reactive TCRß or
TCR chains with a wide variety of TCR or TCR chains preserves
reactivity (51, 55). Whereas the experiments presented here draw
attention to the importance of the TCR chain junctional region in
prenyl pyrophosphate Ag reactivity, we wanted to determine whether
expression of the TCR chain alone was sufficient to confer
reactivity. We contransfected the cDNA encoding the V2 TCR chain
from the EPP-reactive V2/W/J1.2 with the cDNA encoding the V1
TCR chain from an irrelevant TCR (SPF7) into 31.13, a
TCR- mutant of Jurkat T cells. The resulting
transfectant, termed V2/W/J1.2-V1, was fully capable of
responding to anti-TCR mAbs by releasing IL-2, but was not reactive
to the prenyl pyrophosphate Ag analogue EPP (Fig. 7). This transfectant also failed to
react with other prenyl pyrophosphate Ags or with Ags in mycobacterial
supernatants (data not shown). Thus, surface expression of V2 paired
with V1 is insufficient for recognition of prenyl pyrophosphate Ags
and is consistent with the previous observations from our laboratory
and others that only T cells bearing the V2V2 TCR chain pair
react to such Ags (7, 9, 32, 40).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
recognize Ag in a way that is more like Igs than like TCRß. In the
mouse, T cells directly recognize the MHC class II molecule
I-Ek and the nonclassical MHC molecules T10 and T22
without peptide dependence. Site-directed mutagenesis of the
I-Ek MHC molecule showed that T cells recognized MHC
residues outside the peptide binding groove (56, 57). Moreover,
T cells recognized native, unprocessed herpes simplex virus
glycoprotein I even when bound to plastic without professional APC (58, 59). Analysis of CDR3 length distributions of TCR chains indicates
that they are more similar to those of IgH than to TCR and TCRß
chains (60). Finally, recent crystallographic studies have shown that
TCR V domains model more closely on VH than on TCR V
or Vß (61).
In this report we demonstrate that a change in the junctional (N
and J) region of the TCR chain has a profound effect on prenyl
pyrophosphate Ag recognition. The substitution of an entire TCR
junctional region for another (changing the wild-type V2/W/J1.2
to V2/GN/J1.3) resulted in the loss of reactivity to EPP and IPP,
but preserved totally reactivity to another phosphate Ag in the
partially purified mycobacterial supernatant containing prenyl
pyrophosphate Ags (Figs. 3
and 4). Moreover, a seemingly minor
substitution of one N nucleotide region-encoded sequence for another
within the TCR CDR3 loop destroyed reactivity to EPP and to the
mycobacterial supernatant (Fig. 6). These unpredicted results suggest
that the TCR junctional region may be important in determining the
fine specificity of V2V2 TCR-mediated recognition of prenyl
pyrophosphate Ags. Additional experiments to validate this conclusion
await the purification and identification of the biologically active
phosphate Ag in mycobacterial supernatants capable of activating both
the wild-type V2/W/J1.2 and the chimeric V2/GN/J1.3
transfectants. These experiments do not rule out roles for other
TCR CDR regions in prenyl pyrophosphate Ag recognition. Nonetheless,
the loss of reactivity to known prenyl pyrophosphate Ags such as EPP
and IPP by this chimeric transfectant differing only in TCR
junctional sequence argues against V gene-mediated superantigen-like
recognition of prenyl pyrophosphate Ags. Recognition of the prenyl
pyrophosphate Ags and their analogues more closely resembles that of
Ig-mediated recognition of the phosphate-containing hapten
phosphorylcholine, which involves critical contacts with CDR3-encoded
segments as well as V segment-encoded residues and can be abrogated
by point mutations of key CDR3 H and L residues (62, 63).
The nature of the interaction of the TCR and prenyl
pyrophosphate Ags is largely unexplored compared with the extensive
knowledge available regarding TCRß/peptide interactions. Based on
transfection experiments we know that the V2V2 TCR is required
for reactivity to prenyl pyrophosphate Ags, and that responding T cell
clones are junctionally diverse. This diversity is primarily due to N
nucleotide additions and to V-J nibbling. However, germline-encoded J
region sequences also contribute to junctional diversity, and in the
current example, either J1.2 or J1.3 is permitted in prenyl
pyrophosphate-reactive T cell clones. Moreover, there exist V2V2
clones that are nonreactive to prenyl pyrophosphate Ags, and
V2J1.3-expressing T cell clones tend to be over-represented in
this nonreactive subset (7, 9, 40). V2V2 TCRs rearranging to
J1.3 are rare in adult peripheral blood but are more common in fetal
tissues and in the thymus (7, 9, 64). Whereas culture conditions may
select against V2V2 T cells expressing J1.3, a more likely
explanation for their rare occurrence in human peripheral blood is that
V2V2 T cells expressing the J1.3 sequence are not expanded by
antigenic exposure in vivo. This lack of in vivo expansion may be due
to this relative lack of reactivity of the TCR to certain Ags to which
humans are routinely exposed early in development. The Ags important
for in vivo reactivity have not been determined directly. However, we
speculate that one such set of selecting Ags are prenyl pyrophosphates,
since they are secreted bacterial products produced by a wide variety
of micro-organisms, and V2V2 T cell expansions are associated
with many human infections (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 65).
Previous work shows (34) (C. T. Morita, unpublished observations)
that prenyl pyrophosphate Ag-reactive, junctionally diverse V2V2
T cell clones react to various prenyl pyrophosphate Ags with the same
hierarchy of responsiveness. Thus, a particular prenyl pyrophosphate Ag
that elicits the strongest response from one V2V2 T cell clone on
a molar basis typically elicits the strongest response from all other
V2V2 T cell clones tested. If V2V2 T cell clones in vivo
truly recognize all prenyl pyrophosphate Ags in the same way, why is
there such enormous junctional diversity? This part of the puzzle
remains unexplained, but may point to still undiscovered Ags or
Ag-presenting molecules that are recognized by some of these
junctionally diverse V2V2 TCRs but not by others. Crystal
structures of Ag- TCR complexes will be needed to determine how
the V2V2 junctional regions contact prenyl pyrophosphate Ags.
However, this report demonstrates a crucial role for the TCR
junctional region in the recognition of prenyl pyrophosphate Ags,
predicting that direct CDR3 contact with prenyl pyrophosphate Ags
should occur. Moreover, this finding distinguishes recognition of such
nonpeptide Ags from that of protein superantigens whose recognition is
solely determined by V gene-encoded residues.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack F. Bukowski, Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115.
3 Abbreviations used in this paper: IPP, isopentenyl pyrophosphate; EPP, ethyl pyrophosphate; MEP, monoethyl phosphate; CDR, complementarity determining region.
Received for publication November 21, 1997. Accepted for publication February 26, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-cell receptor. Immunol. Rev. 120:137.[Medline]
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