By
From the * Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy,
Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston,
Massachusetts 02115; the Department of Molecular Biology and Skaggs Institute of Chemical
Biology, Scripps Research Institute, La Jolla, California 92037; and the § Division of Dermatology,
University of California Los Angeles School of Medicine, Los Angeles, California 90095
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Abstract |
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The T cell antigen receptor (TCR) mediates recognition of peptide antigens bound in the
groove of major histocompatibility complex (MHC) molecules. This dual recognition is mediated by the complementarity-determining residue (CDR) loops of the and
chains of a single TCR which contact exposed residues of the peptide antigen and amino acids along the
MHC
helices. The recent description of T cells that recognize hydrophobic microbial lipid antigens has challenged immunologists to explain, in molecular terms, the nature of this interaction. Structural studies on the murine CD1d1 molecule revealed an electrostatically neutral
putative antigen-binding groove beneath the CD1
helices. Here, we demonstrate that
/
TCRs, when transferred into TCR-deficient recipient cells, confer specificity for both the foreign lipid antigen and CD1 isoform. Sequence analysis of a panel of CD1-restricted, lipid-specific TCRs reveals the incorporation of template-independent N nucleotides that encode diverse sequences and frequent charged basic residues at the V(D)J junctions. These sequences
permit a model for recognition in which the TCR CDR3 loops containing charged residues
project between the CD1
helices, contacting the lipid antigen hydrophilic head moieties as
well as adjacent CD1 residues in a manner that explains antigen specificity and CD1 restriction.
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Introduction |
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Acentral event in the recognition of microbial pathogens is the interaction of specific TCRs with their
ligands (1). In most cases, these ligands are perceived to
be complexes between MHC molecules and peptide antigens. Recently, we identified human /
T cells that respond to lipid and glycolipid antigens from mycobacteria
and that are restricted by CD1a, b, or c. These three CD1
isoforms are distantly related to MHC molecules and are
expressed on professional APCs, including B cells, macrophages, and dendritic cells, as well as transiently on thymocytes during development (4). The expression of
CD1a, b, and c on APCs and the bacterial origin of the
lipid antigens they present suggest a role for lipid-specific, CD1-restricted T cells in host defense.
The appreciation that human T cells can detect lipid antigens and be restricted by molecules other than those encoded in the MHC raises fundamental questions about how nonpeptide antigens and CD1 molecules are recognized. The atomic structure of mouse CD1d1 resembles class I MHC molecules in overall topography, but in place of the peptide-binding groove of MHC, CD1d1 possesses an antigen-binding super domain composed of a hydrophobic and nonpolar cavity potentially capable of binding the acyl chains of lipid antigens (9). This finding suggests that CD1 molecules may have evolved to function as lipid-binding antigen-presenting molecules. In support of this hypothesis, recent data demonstrate a direct interaction between purified glycolipid antigens and CD1b (10) and indicate that glycosylphosphatidylinositol is a self lipid bound by murine CD1d1 (11).
The recognition of human CD1d and mouse CD1d1 has
been linked to a population of T cells expressing an invariant, germline-encoded V24-J
18 TCR
chain paired
primarily with V
11 TCR
chains (12, 13). T cells bearing the invariant V
24-J
18 TCR
chain are stimulated
by CD1d+ APCs, suggesting that the TCR on these cells
may interact directly with CD1d, or with CD1d containing bound self-lipids. In contrast to CD1d-restricted T
cells, which respond to APCs in the absence of a foreign
antigen, the CD1a-, b-, and c-restricted T cells examined
in this report only respond in the presence of foreign mycobacterial lipid antigens. Nothing is known about the
TCRs that recognize CD1a-, b-, and c-restricted foreign
lipid antigens. Thus far, it has not been possible to visualize
the molecular basis for recognition of long chain mycolic
acids and glycolipids like lipoarbinomannan. No data are
available as to whether only invariant TCRs recognize
these foreign lipid antigens and no model has been proposed for how the TCR might interact with an amphipathic lipid antigen-CD1 complex.
To address these questions, we have cloned the TCRs
from a panel of CD1a-, b-, or c-restricted, lipid antigen-
specific T cells. By gene transfer, we demonstrate that the
TCR confers specificity for both foreign lipid antigen and
CD1 isoform. Sequence analyses revealed notable diversity
in TCR gene segment usage, indicating that invariant
TCRs that recognize CD1d are not predictive of the
TCRs against microbial lipid antigens. Importantly, frequent usage of basic amino acids in the complementarity-determining residue (CDR)31 regions suggests a model for
the interaction of TCR with lipid-CD1 complexes in
which these TCR loops project directly between the CD1
helices and mediate electrostatic interactions with the polar functions of amphipathic lipid and glycolipid
antigens.
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Materials and Methods |
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Cell Lines and mAbs.
The T cell lines used in this study, DN1, CD8-1, CD8-2, DN.POTT, and LDN5, have been described previously (4, 6, 14). T cells were cultured in 24-well Linbro® tissue culture plates (ICN Biomedicals, Inc.) in RPMI 1640 supplemented with 10% FCS, Hepes, L-glutamine, essential and nonessential amino acids, sodium pyruvate, beta-mercaptoethanol, and penicillin/streptomycin (complete medium) and containing 1.5 nM recombinant human IL-2 (rhIL-2) (Ajinomoto Co.). T cell lines were restimulated every 2 wk with CD1+ monocytes and a chloroform/methanol extract of Mycobacterium tuberculosis H37Ra (prepared from a 200 mg dry bacteria/ml suspension; Difco Laboratories) at a 1:5,000 dilution. CD1+ monocytes were prepared as described previously (15). In brief, adherent cells from random donor Leukopacks were cultured for 3 d in complete medium containing 300 U/ml GM-CSF (Immunex Corp.) and 200 U/ml IL-4 (gift of Schering Corp.). J.RT3-T3.5 cells, derivatives of Jurkat cells that have defective endogenous TCRPreparation of Lipid Antigens.
The lipids recognized by the CD8-1 and CD8-2 T cell lines were partially purified as follows. M. tuberculosis H37Ra (Difco Laboratories) was sonicated in PBS and extracted with chloroform/methanol in a 2:1 ratio. The organic phase was recovered, dried by rotary evaporation, and resuspended in chloroform. The organic extract was then fractionated using silica gel chromatography. The organic extract equivalent of 10 mg dry bacteria was loaded in chloroform onto a 1 g silica gel (Selecto Scientific) column. The column was then eluted with a step gradient of methanol in chloroform from 0 to 100% methanol in 10% increments. The lipid(s) recognized by CD8-1 were found to fractionate predominantly in the 60:40 chloroform/methanol fraction and the lipid(s) recognized by CD8-2 were found predominantly in the 90:10 chloroform/ methanol fraction (hereafter referred to as silica fraction 60:40 and silica fraction 90:10, respectively; reference 19a). To isolate mycolic acids from M. tuberculosis H37Ra, dry bacteria were suspended in chloroform/methanol in a 2:1 ratio to extract the majority of the lipids from the cell wall. The delipidated cell walls were pelleted and dried. The pellet was saponified with a methanol/potassium hydroxide solution to release mycolic acids, which were then extracted with hexane. Mycolic acids were then precipitated with ether/ethanol and dissolved in chloroform. The presence of alpha, methoxy, and keto mycolic acids was confirmed by thin-layer chromatography (data not shown).T Cell Cloning.
T cell clones were isolated from cell lines by limiting dilution. T cells were plated at 0.3, 1, or 5 cells/well in 96-well round-bottomed microtiter plates. Normal human PBMC (105/well) and a 1:1 mixture of two EBV-transformed human B cell lines (5 × 104/well) were irradiated (5,000 rad) and added as feeder cells. PHA-P (Difco Laboratories) was added to a final concentration of 1:4,000 in a total volume of 150 µl/well in complete medium containing 1.5 nM rhIL-2. Plates were fed after 7 d of culture at 37°C and then every 3-4 d thereafter. Clones were expanded by restimulation with PHA and feeder cells every 2-3 wk.T Cell Proliferation Assays.
T cells (5-50 × 103/well) were cultured in triplicate in 96-well flat-bottomed microtiter plates with or without irradiated (5,000 rad) CD1+ monocytes (5 × 104/well) in the presence or absence of antigen. To assess CD1 restriction, purified mAbs specific for CD1a, b, or c were added at a final concentration of 20 µg/ml. Cells were cultured for 3 d at 37°C, pulsed with [3H]thymidine (1 µCi/well, 6.7 Ci/mmol, New England Nuclear) and incubated for 5-6 h. The plates were harvested on a Tomtec 96-well plate harvester (Wallac, Inc.) and thymidine incorporation was measured with a Betaplate liquid scintillation counter (Wallac Inc.).Inverse PCR.
Inverse PCR was used to determine the TCRTCR Cloning.
To isolate full-length cDNAs encoding the TCRs, we designed VTCR Transfection.
Flow Cytometry.
Transfectants were analyzed for cell surface expression of TCR-CD3 complexes as follows. Cells were incubated for 45 min on ice in the presence of 20 µg/ml SPVT3b (anti-CD3T Cell Transfectant Stimulation Assay.
J.RT3-T3.5/TCR-TCR-CD1b Modeling.
Alignment of amino acid sequences was performed using the programs PILEUP and BESTFIT, part of the Wisconsin Package Version 9.1 (Genetics Computer Group). The primary structure of human CD1b was aligned with the murine CD1d1 molecule (46.9% identity). Residues differing between proteins were mutated using the program O (24), and the conformation for different side chains was assigned based on the rotamer library and the main chain conformation (25). The model was subjected to 250 steps of energy minimization as implemented in the program X-PLOR (26). Three cycles carried out after assignment of different initial velocities resulted in virtually identical models. Bad contacts between side chains were checked manually. The sequences of the DN1 ![]() |
Results |
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Five
lipid-specific human T cell lines have been characterized as
restricted by CD1a (CD8-2), CD1b (DN1, DN.POTT,
LDN5), and CD1c (CD8-1) (Table I) (4, 6, 14). To study
the nature of the TCR in recognition of CD1-presented
lipid antigens, several of these T cell lines were cloned by
limiting dilution, and the specificities of the T cell clones
were confirmed in proliferation assays using purified lipid/
glycolipid antigens and CD1+ monocyte APCs. The CD1
restriction of these clones was confirmed by blocking proliferation with mAbs specific for CD1a, b, or c. A clone
isolated from the CD8-2 T cell line (designated CD8-2.1) proliferated when cultured with CD1+ monocytes and the
lipid fraction of M. tuberculosis strain H37Ra designated silica fraction 90:10 (see Materials and Methods). Proliferation was blocked by 96% by the inclusion of the anti-CD1a mAb 10H3.9, but not by control mAb P3, anti-CD1b
mAb BCD1b3.1, or anti-CD1c mAb F10/21A3.8 (Fig. 1
A). Similarly, clones isolated by limiting dilution from the
DN1 (designated DN1.F9, Fig. 1 B) and CD8-1 (designated CD8-1.A2.3, Fig. 1 C) T cell lines proliferated in the
presence of M. tuberculosis strain H37Ra mycolic acids or
H37Ra-derived silica fraction 60:40, respectively. Proliferation of DN1.F9 cells was blocked by 57% with anti-CD1b, but only by 1-24% by control or anti-CD1a or c
mAbs, whereas proliferation of CD8-1.A2.3 cells was
blocked by 88% with anti-CD1c mAb, but not by control
or anti-CD1a or b mAbs. Thus, the specificity of these T
cell clones matches that of the published parent T cell lines
shown in Table I, and, consequently, they were used as sources of RNA for TCR molecular cloning. Repeated T
cell cloning attempts with DN.POTT and LDN5 cell lines
failed to yield clones that expanded adequately for complete analysis. Consequently, RNA was extracted from
these two long-term homogeneous, but uncloned, T cell
lines for TCR analysis. The specificity of the transfectants derived was assessed to assure that they displayed the specificity of the parental cell lines, indicating that the correct
antigen-specific TCR and
chain pairs had been isolated.
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To identify the TCR gene usage of these T cell lines and
clones, we used inverse PCR analysis, which allows cloning of any TCR without prior knowledge of V or J gene
segment usage. Double-stranded cDNA was synthesized
from total cellular RNA and circularized by blunt-ended
ligation. The circular cDNAs were then used as templates
in PCR reactions with pairs of C- or C
-specific primers oriented in opposite directions. The resulting amplicons
thus included the TCR V, N/D/N, and J regions of the
TCR genes, which allowed the identification of TCR
gene segment usage. The inverse PCR products obtained
from the CD1-restricted T cell cDNAs were of the expected size (~700 bp), and these products were cloned into
pBluescript II and sequenced.
Although /
TCRs
clearly interact with peptide-MHC complexes, it is not
known what role the TCR plays in the recognition of lipid
antigens and CD1 molecules. To test whether
/
TCRs mediate lipid antigen-specific, CD1-restricted recognition, we cloned full-length cDNAs encoding the TCR
and
chains from the DN1.F9, CD8-1.A2.3, and CD8-2.1 T cell clones. The TCR gene segment usage identified
by inverse PCR analysis allowed design of V gene-specific
5' untranslated region (UTR) primers for the appropriate
V
and V
genes, identified for each clone, together with
C
or C
3' UTR primers. The TCR-
and TCR-
cDNA PCR products were cloned into the pREP7 (
chains) and pREP9 (
chains) episomal mammalian expression vectors. TCR-
-deficient Jurkat T cells (J.RT3-T3.5) were cotransfected with the TCR-
and TCR-
cDNAs
from DN1, CD8-1, or CD8-2, and selected for 3-4 wk in
medium supplemented with G418 and Hygromycin B,
yielding transfectant cell lines designated DN1/J.RT3,
CD8-1/J.RT3, and CD8-2/J.RT3, respectively. Flow cytometric analysis of the resulting T cell transfectant lines
showed that DN1/J.RT3 was 48% CD3+ (with an MFI of
180), CD8-1/J.RT3 was 80% CD3+ (with an MFI of 264),
and CD8-2/J.RT3 was 60% CD3+ (with an MFI of 219),
whereas mock-transfected J.RT3-T3.5 cells were essentially CD3
(6% positive with an MFI of 60) (Fig. 2, A-D),
indicating that transfected TCR genes were successfully
expressed in association with endogenous CD3.
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To test the TCR transfectants for recognition of lipid/glycolipid antigens and CD1, DN1/J.RT3, CD8-1/J.RT3, and CD8-2/J.RT3, cells were cultured with CD1+ monocytes and lipid antigens and IL-2 production was measured as an index of antigen-specific activation. CD8-2/J.RT3 cells produced IL-2 in a dose-dependent manner when cultured with the appropriate lipid fraction of M. tuberculosis H37Ra (silica fraction 90:10; see Materials and Methods), but not with other lipid preparations that did not contain the specific antigen recognized by CD8-2 (silica fraction 60:40 or purified mycolic acids) (Fig. 3 top). In contrast, DN1/J.RT3 and CD8-1/J.RT3 cells were specifically activated to secrete IL-2 only in the presence of purified M. tuberculosis mycolic acids or silica fraction 60:40, respectively (Fig. 3, middle and bottom). Thus, the lipid antigen specificity of these TCR transfectants matched precisely the antigen specificities of the original T cell lines. The sensitivity of the DN1/J.RT3 cells for antigen was ~50-fold lower than the sensitivities of CD8-1/J.RT3 and CD8-2/J.RT3 cells for their respective antigens. It is not clear if this difference reflects variability in TCR expression levels, antigen uptake or loading efficiency, affinity of the TCR for different ligands, or some other parameter.
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Similarly, full-length TCR- and TCR-
cDNAs were
cloned from the LDN5 cell line using the sequence information obtained by inverse PCR analysis. Transfection of
the LDN5
TCR into J.RT3-T3.5 cells conferred the
ability to respond to purified Mycobacterium phlei glucose
monomycolate but not to other lipid or glycolipid antigens
(data not shown). Although the TCR from DN.POTT
was not reconstituted in J.RT3-T3.5 cells, 6/6 inverse
PCR products representing in-frame TCR-
and 6/6
PCR products representing in-frame TCR-
transcripts
were identical in sequence to those shown in Table II, suggesting that the DN.POTT T cell line is essentially clonal and providing primary sequence data on its TCR (data not
shown).
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Since J.RT3-T3.5 cells express an endogenous TCR chain with which the transfected TCR
chains could potentially form a second functional heterodimer, we made
control J.RT3 cells transfected with only the TCR
chains of each of the TCRs studied. Although TCR
transfectants typically expressed levels of TCR at the cell surface similar to TCR-
/
transfectants and could be
stimulated with anti-CD3, no stimulation of IL-2 production in response to CD1+ APCs with or without lipid antigens was observed (data not shown). Thus, both TCR
and
chains from the lipid/CD1-specific T cell lines were
required to reconstitute a TCR capable of lipid-specific recognition.
The CD1 dependence of the activation of the transfectants was determined using blocking mAbs specific for CD1a, b, or c. IL-2 production by CD8-2/J.RT3 transfectant cells was blocked by 95% with mAb 10H3.9.3 (anti-CD1a), but was slightly increased by mAbs BCD1b3.1 (anti-CD1b) and F10/ 21A3.1 (anti-CD1c). Similarly, the activation of DN1/J.RT3 and CD8-1/J.RT3 transfectants was specifically blocked by anti-CD1b and anti-CD1c, respectively, but not by mAbs to the other two CD1 isoforms tested (Fig. 4, A-C). Therefore, reconstitution of the TCRs from three different CD1- restricted T cells in TCR-deficient J.RT3-T3.5 cells confers recognition of both the specific antigens and the particular CD1 isoforms the original T cells were defined to recognize. These results support a model in which the TCR interacts with both lipid/glycolipid antigen and CD1 analogous to conventional TCR-MHC-peptide interactions.
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Having confirmed that /
TCRs mediate lipid-specific, CD1-restricted recognition,
and having defined that the specific TCR
/
pairs cloned
from a panel of T cell lines were the correct ones for recognition, we next analyzed the TCR sequences to gain insight into the nature of TCR diversity for lipid-specific and CD1-restricted T cells. The DNA sequences were aligned
with known TCR V and J segments using BLAST homology searches (34). Two of the TCRs, those from CD8-2
and DN.POTT, expressed the AV16S1 gene, and the
other TCRs contained three different V
genes, AV1S3, AV8S2, and AV3S1 (Table II). Five different J
segments
were detected in the TCRs from these cells (AJ17, AJ34,
AJ31, AJ57, and AJ9). The TCR V
genes were similarly
diverse, with BV2S1, BV9S1, BV6S1, BV5S1, and BV7S1
each represented once (Table II). However, significantly less diversity was observed in J
usage. Of the 13 functional human J
gene segments, only J
segments BJ2S1 or
BJ2S7 were utilized by the 5 T cell lines. The deduced
amino acid sequences of the TCR V-N/D/N-J junctions
displayed in Table II indicate that the CDR3 regions of
these TCRs have typical lengths (9-13 residues in CDR3
and 8-13 residues in CDR3
) and heterogeneous peptide sequences. Together, the combinatorial diversity of TCR
V
, J
, and V
segment usage and the junctional diversity
of CDR3-encoded residues make it clear that, unlike the
invariant TCR-
genes of NKT cell recognition of CD1d,
those of foreign lipid-specific, CD1a-, b-, or c-restricted
TCR are diverse receptors similar to those of peptide-specific, MHC-restricted TCRs. Despite this general diversity,
close scrutiny revealed an overrepresentation of basic residues, particularly arginine, concentrated near the NH2 terminal ends of the CDR3 regions. These basic residues appear in the
chain (CD8-2), in the
chain (DN1), or in
both
and
chains (CD8-1 and DN.POTT) of the
/
TCR pairs (Table II), suggesting an important role in the
specific recognition mediated by all of these TCRs (see below).
Given the overall structural similarity between MHC and the mouse
CD1d1 crystal structures, together with evidence for lipid binding to human CD1b, we reasoned that lipid-CD1
complexes may form a TCR ligand analogous to peptide-
MHC. Furthermore, our analysis of CD1-restricted TCR
sequences indicates that they are comparable in primary
structure to peptide-MHC-specific TCRs, using the same
gene elements and having CDR3 lengths of comparable size to those found in MHC-restricted TCRs. Therefore,
we modeled the DN1 TCR variable domains and human
CD1b based on the known structures of other /
TCRs
and the mouse CD1d1 protein, respectively. We then
modeled the DN1 TCR as a complex with human CD1b
using the TCR/MHC/peptide ternary crystal structures to
orient the molecules together (Fig. 5). This model suggests
that the DN1 TCR could interact with CD1b in a manner
that is similar to that of TCR
/
with peptide-MHC
complexes. Strikingly, this would result in the positioning
of the TCR CDR3 loops over the center of the putative CD1 antigen-binding site that is flanked by the CD1
helices. Such an orientation would allow the basic residues in
the DN1 TCR
chain CDR3 region to project into the
central region of the groove that would provide direct interactions with exposed portions of a bound lipid or with
the exposed carbohydrate of bound glycolipids. In particular, if the lipid antigen hydrophobic aliphatic chains are
embedded in the electrostatically neutral CD1 pockets, the
hydrophilic end of the lipid (or glycolipid) might be located between the CD1
helices. This would orient the
TCR CDR3 residues to interact with segments of the
CD1
helices and the charged CDR3 residues would
project between the CD1
helices in position to contact
the polar head of the lipid (or glycolipid) (Fig. 5).
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Discussion |
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That T cells bearing /
TCR mediate lipid and glycolipid-specific, CD1a-, b-, or c-restricted recognition
raised questions about the role of the
/
TCR in this
process. Previously, the
/
TCR was thought to interact
solely with MHC class I or II molecules complexed with
antigenic peptides or protein superantigens. Here we have
demonstrated that the TCRs from a panel of lipid/CD1- specific T cells mediate the ability of those T cells to discriminate between foreign lipid antigens and CD1 isoforms.
The recent crystal structure of mouse CD1d1 revealed the
presence of a hydrophobic binding cavity potentially capable of accommodating the hydrophobic aliphatic portions
of a lipid antigen (35). Together with these structural data
and the recent evidence that glycolipid antigens can bind
directly to human CD1b in vitro (10), our findings suggest
that lipids and CD1 molecules form complexes analogous to peptide-MHC complexes that are recognized together
by a single TCR.
Recognition of lipid-CD1 complexes by the /
TCR
greatly expands the universe of foreign antigens recognized
by T cells and reveals that previous assumptions that
/
TCRs were exclusively peptide/MHC-specific were not
entirely correct. Although the relative frequencies of CD1-restricted versus MHC-restricted T cells are unknown, the
prominent expression of CD1a, b, and c on many professional APCs suggests that recognition of this lineage of antigen-presenting molecules may be substantial. Here, the
TCRs were derived from both the major CD8+ and relatively minor CD4
CD8
T cell pools that contain foreign
lipid/CD1-specific T cells. We have also recently demonstrated that
/
T cells can recognize CD1c via a TCR-
dependent mechanism (Spada, F., E.P. Grant, D. Leslie,
and M.B. Brenner, unpublished observations), indicating the ability of both TCR types to interact with CD1.
The sequence analysis of a cross-section of CD1-
restricted T cells shows dramatic heterogeneity in V, V
,
and J
gene usage among the TCRs. This diversity is in
marked contrast to human CD1d-restricted T cells, which
use an invariant germline-encoded TCR
chain composed
of a precise AV24S1-AJ18 rearrangement together with a
restricted pattern of V
chains (36), and to murine CD1d1-restricted NK T cells which use a similar TCR (37). These
CD1d-specific T cells recognize CD1d in the absence of
exogenously supplied lipid antigens, and it is not clear if
they respond to empty CD1d molecules or to CD1d complexed to endogenous lipids/glycolipids. In the analysis presented here, three CD1b-restricted TCRs (DN1, DN.POTT,
and LDN5) are described that use three different V
and V
genes and have sequence diversity in both TCR-
and
-
CDR3 regions. Thus, the recognition of exogenous
lipid antigens with CD1b is more like peptide-MHC recognition than the response to CD1d. By extension, we expect that T cells specific for CD1a- and CD1c-restricted
lipids will also have significantly greater diversity than has
been observed for CD1d-specific T cells. This hypothesis is
supported by the presence of template-independent N nucleotides in the TCR-
and -
sequences of CD8-2
(CD1a-restricted) and CD8-1 (CD1c-restricted) TCRs.
Despite the diversity that we observed in TCR usage,
we noted the frequent coding of basic residues in CDR3
and CDR3
, primarily as a result of N nucleotide additions. If the hydrophobic antigen-binding pocket of CD1
binds to the hydrophobic acyl portions of a lipid antigen,
the polar phosphate, carboxylic acid, or carbohydrate regions of the antigens would be exposed at the opening in
the CD1 groove between the CD1
helices to facilitate
direct contacts with the TCR. We speculate that in the case
of the mycolic acid-specific TCRs (DN1 and DN.POTT)
the positively charged residues in the TCR CDR3 regions
are positioned to interact with negatively charged lipid
antigen head groups that would be exposed between the
CD1
helices. It is noteworthy that the one TCR described here that is specific for an antigen which does not
have a negatively charged head group, LDN5, lacks positively charged amino acids in the CDR3 regions. We generated a molecular model of the DN1 TCR variable domains together with a model of the CD1b
1 and
2
domains based on the crystal structures of TCR-MHC-
peptide complexes (27, 28) and the murine CD1d1 molecule (9) in order to visualize the hypothetical mode of interaction of these proteins (Fig. 5 A). This model suggests
that CD1-restricted TCRs could interact with CD1 in a
way analogous to TCR-MHC. Importantly, this model readily positions the basic residues R97 and R98 in the
DN1 CDR3
loop in a location favorable for direct interaction with any moieties protruding from the CD1b hydrophobic pockets and positioned between the CD1
helices (Fig. 5 B). Given the likely possibility that lipid
binding to CD1 would be primarily through hydrophobic
interactions of acyl chains inside the hydrophobic cavity,
we speculate that the polar regions of the lipid antigens
would be most likely to be exposed at the opening to this
putative antigen-binding site. In the case of mycolic acid,
the most polar region of the molecule consists of a carboxylic acid group and a hydroxyl group. Therefore, one or
both of the arginine residues in the DN1 CDR3
might
directly interact with this region of a bound mycolic acid
molecule, making contacts important for the specificity and
strength of the interaction between TCR and CD1-lipid,
as illustrated schematically in Fig. 5 C. Ultimately, crystallographic studies are required in order to determine whether
this model is correct and to reveal any unexpected differences between the mechanisms by which TCR interacts
with lipid-CD1 and peptide-MHC ligands.
All of the TCRs isolated in this study incorporate either
the BJ2S1 or BJ2S7 gene segments. This finding is also in
contrast to the TCR sequences reported for CD1d-reactive
T cells, which show no obvious bias in J segment usage.
Interestingly, the TCRs expressed by a CD1 directly reactive
jejunal IEL T cell line described by Balk et al. predominantly
used BJ2S1, and a CD1c directly reactive T cell clone isolated from that line expressed BJ2S7 (38). Additionally, two
other CD1a or CD1c directly reactive T cell lines derived in
our laboratories also express BJ2S1 and BJ2S7 (Grant, E.P., S.A. Porcelli, and M.B. Brenner, unpublished observations).
The BJ2S1 and BJ2S7 gene products are quite similar to one
another and are distinguished from the other J
segments by
the motif `EQ(Y/F)F', which is predicted to lie near the
COOH-terminal end of the CDR3
. It is possible that this
portion of the CDR3
mediates important interactions with
CD1a, b, and c molecules themselves, accounting for their
increased incidence in the TCRs analyzed here. Alternatively, these residues may be critical for the positioning of
the CDR loops such that they adopt a conformation suitable for interaction with CD1a, b, or c.
In these analyses, we have examined the receptors from a panel of T cells with diverse specificities for lipid antigens and CD1 isoform restriction. Despite this diversity, we have identified two potentially important TCR structural features that may be involved in the interaction of these TCRs with their lipid-CD1 ligands. We did not observe any clear bias in V gene usage by these TCRs, which testifies to the diversity of the T cell repertoire capable of interacting with nonpolymorphic CD1 molecules and their lipid antigens. Nonetheless, it is possible that a more extensive analysis of T cells with specificity for a single lipid antigen and with a common CD1 isoform restriction will reveal additional antigen-specific or CD1a-, b-, or c-specific biases in V gene usage.
Although the CD1 family of proteins is quite divergent
from MHC at the sequence level, it is now clear that the
two types of antigen-presenting molecules have sufficient
structural similarities to allow the same set of TCR V and J
segments to be combined to form TCR heterodimers that
can interact with two extremely distinct classes of antigens.
Thus, the previous paradigm that /
TCRs are specific
for peptide-MHC complexes must be modified. The TCR transfer studies presented here demonstrate directly that diverse TCR-
/
heterodimers instead mediate the recognition of foreign lipid and glycolipid antigens in the context
of nonpolymorphic CD1a, b, and c antigen-presenting elements. Furthermore, although the CD8-1 and CD8-2 T
cell lines are CD8
/
+, reconstitution of the TCRs in the
J.RT3-T3.5 cell line, which is CD8
/
negative, confers
the ability to productively interact with CD1, indicating
that CD8 coreceptor expression is not essential to recognition of these presenting elements. These findings raise additional questions about CD1-restricted T cells, including
how they are selected during thymic development and
how
/
TCRs achieve specificity for CD1 versus MHC,
given the use of the same families of germline TCR elements. Moreover, the relative frequency of CD1 versus
MHC-reactive T cells during the course of infection and in
autoimmune responses must now be evaluated to appreciate the relevance of these two antigen-presenting systems
in various host responses.
![]() |
Footnotes |
---|
Address correspondence to Michael B. Brenner, M.D., Brigham & Women's Hospital, Smith Bldg., Rm. 552, 75 Francis St., Boston, MA 02115. Phone: 617-525-1000; Fax: 617-525-1010; E-mail: mbrenner{at}rics.bwh.harvard.edu
Received for publication 13 October 1998.
We thank Dr. Masahiko Sugita for critical reading of the manuscript and Dr. D. Branch Moody for helpful discussions.
This work was supported by National Institutes of Health grants AI28973 (M.B. Brenner), CA58896 (I.A. Wilson), AI22553 (R.L. Modlin), AR40312 (R.L. Modlin), and the UNDP/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (IMMLEP) (R.L. Modlin).
Abbreviations used in this paper CDR, complementarity-determining residues; rhIL-2, recombinant human IL-2; UTR, untranslated region.
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