By
From the * Departments of Medicine, Experimental leishmaniasis offers a well characterized model of T helper type 1 cell (Th1)-mediated control of infection by an intracellular organism. Susceptible BALB/c mice aberrantly develop Th2 cells in response to infection and are unable to control parasite dissemination. The
early CD4+ T cell response in these mice is oligoclonal and reflects the expansion of V Leishmania major is a complex intracellular protozoan
parasite of macrophages in the vertebrate host that requires a robust Th1 response for control of infection (reviewed in 1). Requirements for MHC class II molecules, T
cells, IFN- Unlike resistant strains of mice, BALB/c mice develop
aberrant Th2 responses after challenge with L. major and
sustain fatal disseminated disease. The early CD4+ T cell
response is also dominated by expansion of LACK-specific T
cells that express V The L. major model provides an opportunity to test
the plasticity of the wild-type CD4+ T cell repertoire in
susceptible mice. The conserved charge and length of the
peptide-interacting complementarity determining region 3 (CDR3) domain from LACK-reactive T cell hybridomas
suggested the observation that a single parasite antigen
served as the focus of their recognition (2). The constraints
on the entire LACK-reactive repertoire in the intact animal
remain unknown, however. We decided to indirectly test
such constraints by attempting to abrogate activation of
these cells in vivo using altered ligands. We demonstrate substantial protection against disease in this system, suggesting that identification of dominant antigens from organisms
can be used to target pathogenic T cells that mediate progressive disease in a highly specific manner.
Reagents.
Howard Hughes
Medical Institute, University of California San Francisco, San Francisco, California 94143; § World Health Organization Immunology Research and Training Center, Institute of Biochemistry,
University of Lausanne, Epalinges CH-1066, Switzerland;
Cytel Corporation, San Diego,
California 92121; and the ¶ Institute of Molecular and Cellular Pharmacology, University of Nice,
Valbonne 06560, France
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
4/
V
8-bearing T cells in response to a single epitope from the parasite Leishmania homologue of
mammalian RACK1 (LACK) antigen. Interleukin 4 (IL-4) generated by these cells is believed
to direct the subsequent Th2 response. We used T cells from T cell receptor-transgenic mice
expressing such a V
4/V
8 receptor to characterize altered peptide ligands with similar affinity
for I-Ad. Such altered ligands failed to activate IL-4 production from transgenic LACK-specific
T cells or following injection into BALB/c mice. Pretreatment of susceptible mice with altered
peptide ligands substantially altered the course of subsequent infection. The ability to confer a
healer phenotype on otherwise susceptible mice using altered peptides that differed by a single
amino acid suggests limited diversity in the endogenous T cell repertoire recognizing this antigen.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, IL-12, and NOS2 have delineated a pathway
by which CD4+ T cells differentiate into Th1 effector cells
that activate macrophages and confer resistance to the disease. Among mice with I-Ad class II molecules, the CD4+
T cell immune response is focused dominantly on a single
epitope from the parasite LACK1 antigen, which results
in oligoclonal expansion of cells bearing V
4/V
8 heterodimeric TCR in the regional lymph nodes (2, 3). The
dominant nature of this antigen was demonstrated using
TCR-transgenic mice that expressed a monoclonal
T
cell repertoire to this epitope; such mice displayed remarkable control over the organism, despite the absence of T
cells that recognize any parasite-specific antigens other than
the single epitope from LACK (4).
4/V
8 TCR. However, activation of
these cells results in the rapid production of IL-4 that drives
the subsequent Th2 response (5, 6). Deletion of LACK-
reactive cells, either through thymic expression of LACK as
a transgene (7) or superantigen-mediated deletion of V
4-expressing CD4+ T cells (5), attenuated the early production
of IL-4 and promoted the differentiation of Th1 effector
cells that controlled disease in infected BALB/c mice. These
and other data have suggested a model whereby susceptibility to L. major in BALB strain mice is driven by a confluence of factors involving the inherent propensity to produce IL-4 after activation in the setting of an appropriately sized precursor pool of LACK-reactive CD4+ T cells (8).
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Mice.
Female BALB/c, C57BL/6 (The Jackson Laboratory or IFFA Credo), and B10.D2 mice (The Jackson Laboratory) were housed in the University of California San Francisco or University of Lausanne pathogen-free animal facilities and used at 8-10 wk of age. Designated mice were thymectomized at 5 wk of age using standard methods. LACK T cell receptor- specific transgenic (ABLE) mice are TCR-transgenic mice that express a VParasites and Infection.
L. major strains WHOM/IR/MHC Binding Affinities.
I-Ad molecules were affinity purified from cell lysates of A20 lymphoma cells using anti-I-Ad mAb MKD-6 (American Type Culture Collection [ATCC]). Peptides were tested for binding to I-Ad as measured by their capacity to inhibit the binding of 125I-radiolabeled OVA323-336 as previously described (13).Stimulation of ABLE T Cells In Vitro.
Spleen and lymph nodes were harvested from BALB/c ABLE TCR-CT Cell Antagonism Assay.
CD4+ T cells from BALB/c ABLE TCR-CImmune Response in Mice Injected with rLACK Proteins.
Groups of BALB/c mice were injected in the hind footpads with 5, 25, or 50 µg of the designated rLACK protein or 25 µg chicken egg OVA in 50 µl buffer. At various time points, the popliteal lymph nodes were harvested and mRNA purified for analysis of IL-4 transcripts using a semiquantitative reverse transcriptase (RT)-PCR assay as previously described (15). Groups of these treated control or adult thymectomized mice were challenged either 24 h or 10, 20, or 30 d later with either designated rLACK proteins or viable L. major and analyzed by similar methods. BALB/c ABLE-CImmunization with Altered rLACK Proteins.
Nonthymectomized or adult thymectomized BALB/c mice were immunized in the hind footpad with 25 µg purified rLACK proteins or chicken egg OVA in 50 µl of 50 mM Tris/100 mM NaCl, pH 8.0. Mice were infected 24 h later with the designated strains of L. major promastigotes in the left footpad and the course of infection monitored as described above.Cytokine Analysis and Serum IgE Determination.
IL-4 and IFN- ![]() |
Results |
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Using overlapping synthetic peptides and a
panel of T cell hybridomas generated from BALB/c mice
immunized with the recombinant protein, a single I-Ad-
restricted epitope in LACK was localized to amino acids
156-173, comprising the sequence ICFSPSLEHPIVVSGSWD (data not shown). Almost all hybridomas reactive to
LACK expressed a V4/V
8 heterodimeric TCR, although
considerable junctional diversity was apparent. The putative
CDR3 peptide contact domain, however, was generally conserved in length and charge, with a negatively charged
QE or QD motif in the TCR
chain of each of the LACK-reactive hybridomas (3). Similarly, hybridomas established
from the lymph node cells of infected BALB/c mice that
expressed the V
4 TCR contained the QE motif in the
CDR3; one had a charged WD motif at the same position
(2). Such features suggested that a positively charged amino
acid within the LACK antigenic determinant represented a critical TCR contact residue. Based on the use of histidine
and other charged residues at TCR contact points among
peptides binding to I-Ad (16), we mutated the histidine at
position 164 in the wild-type peptide (LACK) to asparagine or lysine, thus creating peptides LACK-N164 and
LACK-K164, respectively.
The relative affinities for MHC class II molecules by LACK and the LACK analogues were tested by assaying their capacities to compete with an I-Ad ligand of known affinity, chicken egg OVA peptide323-336. By this assay, each of the LACK-derived peptides displayed binding affinities for I-Ad in the same nanomolar range as the OVA323-336 reference peptide; if anything, they showed slightly stronger affinities (Table I). Substitution of H164 in the wild-type LACK determinant by N or K did not, therefore, affect its binding affinity for MHC class II molecules.
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ABLE mice express a
transgenic TCR derived from a LACK-reactive V4/V
8
T cell clone that is activated by the LACK156-173 peptide in
the context of I-Ad (4). These mice have been crossed to
BALB/c TCR-C
0 mice, thus creating BALB/c ABLE
TCR-C
0 mice. These mice express a monoclonal
T
cell repertoire consisting exclusively of the LACK-reactive
TCR transgene and were used as a source of T cells, designated ABLE T cells. ABLE T cells proliferated in response
to the LACK wild-type peptide at low concentrations (7 nM)
but not after stimulation with the LACK-N164 or LACK-K164 analogues, even at concentrations up to 4 µM (Fig. 1).
Although ABLE T cells generated both IFN-
and IL-4 in
culture supernatants after incubation with the LACK peptide, neither cytokine was detected after incubation with
the two analogue peptides nor with the irrelevant OVA
peptide that also binds I-Ad (Fig. 1). Thus, a single amino
acid substitution at position 164 in the LACK T cell epitope substantially altered reactivity of the transgenic T cells,
indicating that this amino acid position is likely to be a critical TCR contact residue.
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The LACK-derived peptides were next analyzed in an antagonism assay that was developed to avoid peptide competition at the level of MHC occupancy and thus allow measurements of events mediated by the TCR (13). Spleen cells
from BALB/c TCR-C0 mice were used as APC and were
preincubated with a suboptimal concentration (0.2 µM) of
the wild-type LACK peptide. Washed and irradiated APC
were then incubated with increasing concentrations (0.1-100
µM) of the LACK analogue peptides or the OVA323-336 peptide before ABLE T cells were added and assessed for
their capacity to proliferate and produce cytokines.
LACK-K164 showed dose-dependent inhibition of proliferation to the wild-type peptide; 50% inhibition occurred
at a concentration of 10 µM of the analogue peptide (Fig. 2).
At similar concentrations, the peptide also inhibited IL-4
and IFN- production. LACK-N164 inhibited the proliferation of ABLE T cells only at very high concentrations
(>100 µM). The production of IL-4 was inhibited comparably to the LACK-K164 peptide, but IFN-
production
was inhibited consistently less by LACK-N164 in multiple
assays. The unrelated OVA323-336 peptide displayed no inhibitory activity. Thus, in the presence of otherwise stimulatory amounts of the wild-type LACK peptide, the two analogue peptides behaved as TCR antagonists. Of the TCR-mediated functions tested, LACK-N164 preferentially inhibited IL-4 production by ABLE T cells, whereas the capacity to proliferate and produce IFN-
was less affected;
LACK-K164 was more global in its inhibitory capacities.
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The same amino acid substitutions were introduced
into the full length rLACK protein by site-directed mutagenesis, creating rLACK-N164 and rLACK-K164 altered
proteins. When tested in vitro for its capacity to stimulate
ABLE T cells, the rLACK protein stimulated proliferation
and IL-4 and IFN- production at molar concentrations
comparable to those of the wild-type LACK peptide. In
contrast, the altered rLACK proteins, as their peptide counterparts, did not stimulate proliferation or measurable cytokine production over a wide range of concentrations (data
not shown).
To assess the activity of the rLACK proteins in vivo,
ABLE-C0 mice were injected in the hind footpads with
25 µg of purified rLACK, rLACK-N164, rLACK-K164,
or OVA. After 24 h, the popliteal lymph node cells were
recovered and analyzed using flow cytometry for activation, as assessed by expression of CD69 and enlargement by
light-scattering characteristics. Inoculation of rLACK effectively targeted the transgenic T cells: 80% of V
4+ cells
expressed CD69 (Fig. 3) and forward/side scattering increased significantly (data not shown). The total number
of transgenic T cells actually decreased in the draining
lymph nodes (from 2.9 × 105 after OVA to 1.3 × 105 after
rLACK), consistent with antigen-mediated deletion as previously described in other TCR-transgenic mice (17, 18).
In contrast, V
4+ T cells collected from animals injected
with the rLACK analogues showed CD69 induction and
forward/side scattering indices that were only modestly
greater than those from cells collected from animals injected with the control protein, OVA (Fig. 3). Furthermore, the total number of transgenic T cells in these mice
was not significantly different from the number of transgenic T cells in mice that received OVA (data not shown).
Thus, as assessed by these criteria, the rLACK analogues
did not activate ABLE T cells in vivo in a manner comparable to the cognate LACK protein containing the wild-type epitope.
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Prior experiments demonstrated that the LACK antigen induced prominent IL-4
expression in V4/V
8 CD4+ T cells after injection into
BALB/c mice that reached levels 30-100-fold greater than
after injection of a construct with the I-Ad epitope deleted
(5). Over a 5-50 µg range of rLACK, IL-4 mRNA was
induced 100-fold, whereas no IL-4 mRNA was induced
by either the 41-amino acid LACK deletion mutant or
rLACK-K164 (Fig. 4 A and data not shown). Although a
10-fold induction of IL-4 mRNA was seen after injection
of 5 µg rLACK-N164, no IL-4 mRNA was induced after
injection of 25- or 50-µg doses. None of the LACK derivatives caused induction of IFN-
mRNA under the conditions used. After immunization with CFA, however, each
of the constructs, rLACK, rLACK-K164, and rLACK-N164,
was capable of inducing a proliferative response from subsequently isolated popliteal lymph node CD4+ T cells in
response to their respective LACK156-173 epitopes (stimulation indices increased 10-18-fold; data not shown). No proliferation was induced by any of the LACK epitopes
after immunization with the LACK deletion mutant or
OVA. The 25-µg protein dose was selected for use in subsequent experiments.
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To assess the capacity to alter the response of the endogenous LACK-reactive repertoire, mice were first injected
with 25 µg of the LACK analogue proteins and then, 24 h
later, with either the authentic LACK protein or viable
L. major promastigotes, both shown previously to activate a
brisk IL-4 mRNA response in BALB/c CD4+ T cells (5).
In three separate experiments, prior injection of either analogue protein substantially decreased the subsequent activation of the IL-4 response, consistent with an alteration of
the normal V4/V
8 CD4+ T cell response (Fig. 4 B).
Prior injection of the LACK construct with the deleted
I-Ad epitope or an unrelated I-Ad epitope (OVA) did not
affect the subsequent IL-4 response. Kinetic analysis, in which
the second injection of recombinant LACK was delayed
10, 20, or 30 d after the initial immunization, revealed that
IL-4 nonresponsiveness was maintained for 10-20 d in
mice that had been injected with the mutated LACK analogues but then subsequently recovered. Recovery of IL-4
responsiveness to LACK or L. major injection was ablated
by prior thymectomy (data not shown).
Based on the capacity of LACK analogue proteins to abrogate the early IL-4 response in BALB/c mice, we assessed the ability of immunization to render these mice resistant to progressive disease. Cohorts of mice were immunized once with 25 µg of the various recombinant proteins in the footpad and challenged 24 h later with a lethal infectious dose of wild-type L. major promastigotes of either the IR/173 or LV39 strain.
In three separate experiments with the IR/173 parasite,
the course of disease was significantly attenuated in animals
that received rLACK-N164; no attenuation was seen in
animals that received rLACK-K164 or any of the control
proteins, including the wild-type LACK protein (Fig. 5).
Whereas animals in all of the other groups had to be killed
by week 8, mice that received rLACK-N164 controlled disease up to 12 wk after inoculation, when the experiment
was terminated. Parasitologic control was confirmed by
limiting dilution of tissues that demonstrated a 2-4-log reduction in parasite numbers. Immunologic analysis revealed
a threefold reduction in the number of IL-4-producing
cells in the draining lymph nodes and in serum IgE levels,
whereas the number of IFN--producing cells was similar in all groups.
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Protection was more dramatic using the LV39 strain. Similar to the results using the IR/173 strain, rLACK-N164 but not rLACK-K164 provided lasting protection in a subgroup of nonthymectomized mice (Fig. 6 A). The LACK protein itself provided protection in approximately half of both wild-type and thymectomized BALB/c mice (Fig. 6, A and B). Strikingly, either of the altered LACK proteins, in contrast to the LACK deletion mutant or OVA controls, provided a complete protection in thymectomized mice that was sustained over 100 d (Fig. 6 B). When studied at the conclusion of these experiments, the cure phenotype was associated with attenuation of IL-4 production and control of parasite growth in the footpads that was entirely concordant with the lesion phenotype (data not shown).
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Discussion |
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L. major includes a heterogeneous group of protozoa strains that express some 10,000 proteins from a 35.5-megabase genome (19). Despite this complexity, the early immune response is highly focused on a single epitope from the parasite LACK antigen in mice that express I-Ad MHC class II molecules. As shown here, targeting T cells that recognize this epitope using ligands that differed by a single amino acid from the natural epitope was capable of redirecting an otherwise ineffective immune response with a fatal outcome to a completely protective response with long-term cure. The specificity of the immune intervention suggests limited plasticity in the innate LACK-reactive repertoire in I-Ad-bearing mice, as well as limited ability of Leishmania parasites to mediate progressive infection in such mice in the absence of exuberant LACK recognition.
The mechanisms underlying the dominant recognition
of the LACK epitope remain unclear. Recognition does
not seem related simply to the abundance of the LACK
protein. As compared with other parasite molecules like
the major surface protease gp 63 or the major surface glycolipid LPG, which are present in ~5 × 105 and 3-5 × 106 molecules per organism (20, 21), respectively, LACK
was less abundantly expressed. Quantitation against recombinant standards showed that LACK comprised only
~0.03% of total cellular protein or ~30,000 molecules per
organism (Pingel, S., and R. Locksley, unpublished data).
The LACK epitope displayed in vitro affinity for I-Ad that
was comparable with endogenously eluted I-Ad peptides
(22) and contained a centrally disposed histidine residue,
creating a charged element that has been noted in other peptides that bind this MHC molecule. Presumably, the
dominance of the epitope must result from some confluence of stability and processing of the peptide, the efficiency
of targeting to MHC class II molecules, and/or the size of
the responding T cell repertoire (23). Equally perplexing
is the dominant nature of the V4/V
8 TCR response to
the I-Ad/LACK peptide complex. The convergence of the
immune response on the LACK epitope through use of a
dominant V
/V
-paired TCR has been reported using
other immunogenic peptides (24), suggesting that other antigens or adjuvant-like molecules from live parasites do not
affect this clonal affinity maturation process.
Earlier studies reported the ability to vaccinate susceptible BALB/c mice against L. major using LACK antigen administered in a manner such that LACK-specific Th1 cells
were generated (3, 25). Indeed, LACK-specific Th1 cells were
alone sufficient to establish substantial control over infection
with L. major, demonstrating that immunoreactive LACK
peptide is expressed in vivo at physiologically important
levels (4). Despite the capacity of LACK-specific T cells to
control infection with the parasite in vivo, such T cells are
not required. Thus, BALB/c mice rendered deficient in CD4+ T cells that recognize this dominant epitope, either
through thymic expression and central deletion or by superantigen-mediated deletion of all V4+CD4+ T cells,
were capable of containing L. major infection (3, 5). These
experiments suggested that LACK recognition was required for establishing the susceptible state of BALB/c mice, although neither method directly targeted epitope-specific T
cells. Thus, overexpression of LACK antigen in the thymus
might affect the T cell repertoire in ways other than deletion of LACK-reactive T cells. Similarly, deletion of all V
4+
CD4+ T cells targets cells of additional specificities but unknown contributions to defense against Leishmania.
The ability of peptide ligands that differed at a single
amino acid residue to affect the subsequent course of disease in susceptible BALB/c mice argues strongly for highly
conserved specificity to the Th2-mediating repertoire. Altered ligands are presumed to anergize or functionally alter
discrete populations of T cells by their ability to establish
incomplete signaling through the TCR complex (reviewed
in 26). Modulation of cytokine patterns by altered ligands
has been previously demonstrated (27), although application of this technology to an acute infectious process has
been infrequently examined. Redirection of Th subset differentiation, or immune deviation, has been reported by either variations in antigen dose (28, 29) or through use of
altered peptide ligands (30). For a variety of reasons, we
consider it unlikely that immune deviation can account for
the protection mediated by the altered LACK proteins.
First, over a wide dose range, the LACK peptide caused no
shift in the production of IL-4 and IFN- relative to each
other by the TCR-transgenic ABLE T cells. Second, the altered LACK peptides induced neither proliferation nor
cytokine production by ABLE T cells. Third, the massive
activation of ABLE T cells after injection of LACK was absent after injection of the altered LACK antigens. Finally,
injections of lower doses of LACK or the altered LACK
proteins into BALB/c mice did not induce early production of IFN-
, rather than IL-4, mRNA. We could, therefore, find no evidence for the establishment of a LACK-specific Th1 response that could account for the protection
mediated by the altered LACK analogues.
More likely, protection of susceptible mice was accomplished through tolerance or deletion of LACK-reactive
T cells, a mechanism consistent with previous experimental
findings (5, 7). Both altered LACK proteins antagonized
IL-4 production by transgenic T cells in response to LACK.
When used to immunize BALB/c mice, they abrogated the
early IL-4 response to L. major parasites. Treated mice were
able to control parasite multiplication of the LV39 strain up
to 5 wk; over prolonged periods, and with both the LV39
and IR/173 strains, mice pretreated with the rLACK-N164 protein demonstrated persistent immunity. Thymectomized
BALB/c mice immunized with either rLACK-N164 or
rLACK-K164 were completely protected, a finding consistent with the ability of the altered LACK proteins to abrogate IL-4 production by V4/V
8 CD4+ T cells in these
mice. Presumably, the delayed yet progressive disease in nonthymectomized LV39-infected mice was dependent on new
thymic emigrants. Immunization with the LACK protein
itself conferred protection to some mice. As demonstrated
using the TCR-transgenic mice, this presumably relates to
the capacity of the cognate ligand to mediate peripheral deletion of high-affinity LACK-specific T cells. The observed
differences in the overall grade of protection between the
two strains of L. major were surprising, but such differences
in virulence have been previously described (31, 32), as have
subset responses within cohorts of identically treated mice,
as seen here among rLACK- and rLACK-N164-treated mice (33). Boosting or otherwise optimizing the immunization schedule might have enhanced protection against
the IR/173 strain. The rLACK-N164 ligand, however,
conferred protection against both L. major strains, suggesting that identification of dominant antigens from pathogens
can be used to target disease-producing T cells in a highly
sequence-specific manner.
Previous studies have documented that different T cell clonotypes can respond to the same antigen within a given T cell repertoire. Furthermore, considerable cross-reactivity is an essential feature of the T cell receptor, which assures that pathogenic peptides are efficiently recognized (36). Our results suggest that the endogenous LACK-specific repertoire is highly constrained in its CDR3 recognition domain. Limited plasticity of the endogenous T cell repertoire has been previously noted with certain peptide antigens (37), suggesting that infectious diseases may have contributed to the evolutionary divergence of V region genes. Expression of dominant epitopes by parasites, together with the diversity and size of the responding host T cell repertoire, might greatly affect the outcome of such infections and thus contribute to the highly diverse clinical manifestations of leishmaniasis in human populations.
L. major contains two LACK genes expressed in tandem from the same chromosome. Apart from what is known regarding their mammalian homologues, little is known regarding the biochemical action of these proteins. Aside from the potential vaccine use of this antigen (3, 25), additional study promises to shed much light on the coevolution of host and parasite within the context of the immune system, MHC recognition, and the T cell repertoire. Such studies may have great implications for our understanding of the basis for susceptibility and resistance to infectious diseases.
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Footnotes |
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Address correspondence to Richard M. Locksley, UCSF, Box 0654, C-443, 521 Parnassus Ave., San Francisco, CA 94143. Phone: 415-476-5859; Fax: 415-476-9364; E-mail: locksley{at}medicine.ucsf.edu or Jacques A. Louis, WHO Research and Training Center, University of Lausanne, Epalinges, Switzerland. Phone: 41-21-692-5703; Fax: 41-21-692-5705; E-mail: jacques.louis{at}ib.unil.ch
Received for publication 23 October 1998 and in revised form 12 February 1999.
S. Pingel and P. Launois contributed equally to this work.The authors thank Z.-E. Wang, L. Stowring, C. McArthur, and E. Weider for technical assistance.
This work was supported by grants from the National Institutes of Health (AI26918), the Howard Hughes Medical Institute, the Swiss National Science Foundation, the World Health Organization, and the European Union. D.J. Fowell was supported by a Juvenile Diabetes Foundation International Fellowship.
Abbreviations used in this paper
ABLE, LACK T cell receptor-specific
transgenic;
CDR3, complementarity determining region 3;
LACK, Leishmania homologue of mammalian RACK1;
RT, reverse transcriptase;
TCR-C0, T cell receptor constant region-
-deficient.
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