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Sections of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite these elaborate safeguards, T lymphocytes expressing receptors that can recognize self normally persist within the repertoire (10, 11, 12), and under certain circumstances can contribute to autoimmune pathogenesis (13, 14). In an effort to understand the origins and immune potential of CD8+ T lymphocytes specific for peripheral self Ag, we have focused on a transgenic model in which the influenza virus hemagglutinin (HA)3 (3) gene is expressed by ß cells within the pancreatic islets (15). CD8+ T lymphocytes with high avidity for self MHC-HA peptide become tolerized in the periphery by a mechanism involving their abortive activation in the pancreatic lymph nodes (16, 17). Mice expressing this transgene product demonstrate tolerance of HA even after immunization with influenza virus. Despite such functional tolerance, there exists a residual HA-specific T lymphocyte repertoire that differs significantly from the response by conventional mice, in that it requires high concentrations of HA to detect Ag responsiveness (18). In this report we have focused on the phenotypic and functional capacities of these residual HA-specific CTL.
The simplest explanation for the persistence of low avidity HA-specific
CTL in the repertoire is that they represent naive CTL with low
affinity TCRs that cannot be triggered by naturally occurring
concentrations of Ag. Accordingly, these CTL remain functionally
ignorant of peripherally expressed Ag (19). However, other
mechanisms exist that can down-regulate the avidity of a T lymphocyte
for cognate Ag. A CD8+ lymphocyte with a high
affinity TCR for self can escape deletion by decreasing cell surface
expression of the TCR (8, 20, 21) or by expressing a
second TCR 
-chain (22, 23, 24, 25, 26). This results in decreased
expression of the potentially autoimmune TCR /ß heterodimer, which
spares the cell from thymic or peripheral elimination. In a related
mechanism, it has been shown that decreased expression of the CD8
coreceptor is another way in which the overall avidity of a T
lymphocyte can be reduced and thereby spare self-reactive thymocytes,
although the ability of these CD8low T
lymphocytes to optimally function in the periphery has been questioned
(27, 28, 29). Anergy is another type of mechanism that can
alter the responsive capability of a T lymphocyte.
CD8+ T lymphocytes exposed to supraoptimal doses
of Ag can result in decreased signal transduction through the TCR
complex (12, 30, 31). Importantly, most of what we know
concerning potential mechanisms of peripheral tolerance has resulted
from experiments performed with T lymphocytes from TCR transgenic mice,
which express a TCR of high avidity. None of these potential mechanisms
has been assessed in a conventional, polyclonal T lymphocyte
repertoire, which we address in this report.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The D2 mice were purchased from the breeding colony of The Scripps Research Institute (La Jolla, CA). The InsHA transgenic mice were generated and characterized as previously described (15). All mice were bred and maintained under specific pathogen-free conditions in The Scripps Research Institute’s vivarium. All experimental procedures were conducted in strict accordance with the guidelines laid out in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice used in these experiments were at least 8 wk of age. For adoptive transfer experiments, mice were irradiated (600 rad) before i.v. injection of the specified number of CTL.
Viruses
Influenza virus A/PR/8/34 H1N1 (PR8) was grown in the allantoic cavity of 10- to 11-day-old hen’s eggs. Following isolation, the allantoic fluid was titrated for hemagglutination using chicken RBC. Stocks of recombinant vaccinia virus expressing the Kd-restricted HA epitope 518IYSTVASSL526 (Vacc-KdHA), were provided by Drs. Jack R. Bennink and Jonathan Yewdell from the National Institutes of Health. Vacc-KdHA was propagated on TK- 169 cells at a multiplicity of infection of 0.1. All viruses were stored in 1-ml aliquots at -70°C.
Peptide
The HA peptide (518IYSTVASSL526), which is presented by Kd (32), was synthesized by The Scripps Research Institute core facility using a 430A peptide synthesizer (Applied Biosystems, Foster City, CA). Purity was >85%, as determined by mass spectrometry and reverse phase HPLC analysis on a C18 column (Vydac, Hesperia, CA). Peptide was resuspended in DMSO and stored at -70°C at a concentration of 1 mM.
Generation of KdHA-specific CTL lines and clones
To generate HA-specific CTL, mice were immunized i.p. with 1200 HA units of PR8 in the form of allantoic fluid. Three weeks later, mice were boosted with 108 PFU of Vacc-KdHA in PBS. Mice were rested for at least an additional 3 wk before use in CTL experiments. Single-cell suspensions were prepared from the spleens of immunized mice and seeded into 24-well tissue culture plates at 6 x 106 cells/well in 1 ml of RPMI complete (RPMI, Life Technologies, Gaithersburg, MD) supplemented with 10% (v/v) FCS (Gemini Bio-Products, Calabasas, CA), 2 mM glutamine (Life Technologies), 5 x 10-5 M ß-ME (Sigma, St. Louis, MO), and 50 mg/ml gentamicin (Gemini Bio-Products). To generate APC, single-cell suspensions of spleens from 4- to 6-wk-old D2 mice were irradiated (3000 rad) and then pulsed for 1 h with KdHA peptide at a final concentration of 8 x 10-6 M (for InsHA CTL) or 8 x 10-9 M (for D2 CTL). Restimulating D2 CTL under greater peptide concentration, or InsHA CTL under lower peptide concentrations has been observed to result in either cell death (in the case of D2-derived CTL, as described by Alexander-Miller et al. (33, 34)) or no expansion (in the case of InsHA CTL). Following three washes, 6 x 106 APC in 1 ml of RPMI complete was added to each well containing responder cells. Plates were cultured at 37°C in a humidified incubator with 5% (v/v) CO2. The cytolytic activity of primary CTL lines was assessed on day 6, and responding cultures were restimulated on day 7. CTL were adoptively transferred 10 days after primary stimulation. The InsHA and D2 CTL lines that had been maintained for at least eight passages were cloned by limiting dilution. Briefly, serial dilutions of CTL (from 0.3–30 cells/well) were seeded into 96-well plates with 5 x 105 APC/well in a final volume of 200 µl. Cultures were checked weekly, and wells showing cell growth were sequentially expanded into 48- and 24-well plates before testing for cytolytic activity. An HLA-A2/Kb-restricted CTL clone 12, specific for murine p53261–269, was used as a negative control in tetramer binding experiments (72).
Cytotoxicity assays
Primary CTL cultures were assayed on day 6 for cytolytic activity, and CTL lines and clones were assayed on either day 4 or 5. Target cells were prepared by incubating B10.D2 fibroblast cells at 37°C with 200 µCi of sodium chromate 51 (New England Nuclear, Boston, MA) for 1 h in the presence or the absence of KdHA peptide at the concentrations indicated in the text and figure legends. Target cells were washed and then added to duplicate wells of 96-well round-bottom plates at 1 x 104 cells/well in 100 µl of RPMI complete. Effector CTL were harvested, counted, and added at the appropriate E:T cell ratio in 100 µl of RPMI complete, making a final volume of 200 µl. Plates were incubated at 37°C in a humidified incubator with 5% (v/v) CO2 for 4–5 h. For analysis of perforin-independent lysis by CTL, L1210 (H-2d) Fas+ and Fas- cells (provided by Dr. R. Dutton, Trudeau Institute, Saranac Lake, NY) were used in a 5-h chromium 51 release assay in the presence or the absence of 4 mM EGTA and 3 mM MgCl2, conditions previously shown to inhibit the perforin/granzyme cytotoxic pathway (35). For CD3-redirected lysis experiments, P815 cells were labeled with sodium chromate 51 and incubated with the indicated concentration of 2C11 Ab (anti-CD3) before coincubation with CTL. One hundred microliters of supernatant was removed from each well to assess isotope release using an ICN Isomedic gamma radiation counter (VWR Scientific, San Diego, CA). The percent specific lysis was determined by the formula: percent specific lysis = [(sample release - spontaneous release)/(maximum release - spontaneous release)] x 100. All cytolytic analyses described in this report are from one experiment and are representative of results from a minimum of three experiments.
RNA isolation
Cells of each CTL clone (3–5 x 106)
were harvested on day 4 following restimulation, purified over a Ficoll
gradient, and subjected to poly(A)+ RNA isolation
using either the MicroFast Track or Fast Track 2.0 kit (Invitrogen,
Carlsbad, CA). The RNA was then subjected to RT-PCR analysis.
Superscript II (Life Technologies) RT was used according to the
specifications of the manufacturer to generate cDNA from RNA samples.
For analysis of TCR transcript expression, we used amplimers and
procedures previously described by Zisman et al. (36).
Briefly, 5' amplimers specific for individual V genes were used in
conjunction with a 3' amplimer specific for C in a standard PCR
reaction with an annealing temperature of 55°C using AmpliTaq
polymerase and Q buffer (Qiagen, Valencia, CA) at concentrations
specified by the manufacturer. After 30 cycles, PCR reactions were
analyzed by agarose gel electrophoresis. Amplification products were
excised from the gel and purified using QiaEX II resin (Qiagen) before
cloning into a pCR II sequencing vector (Invitrogen). Plasmids were
purified using Qiagen minipreps and subjected to fluorescent
sequencing, which was conducted by The Scripps Research Institute core
facility on a ABI 377XL automated sequencer (Perkin-Elmer, Foster City,
CA). Sequencing results were analyzed using MacDNAsis software (Hitachi
Software, Hialeah, FL), and identification of the TCR transcripts
was conducted using the Basic Local Alignment Search Tool (internet
address: http://www.ncbi.nlm.nih.gov/BLAST; Ref. 37) in
addition to comparing the sequencing results with published V- and
J-chain sequences (38, 39).
Flow cytometry
Cells of each CTL clone (1 x 106)
were washed, resuspended in 50 µl of FACS buffer (HBSS containing 1%
(w/v) BSA and 0.02% (w/v) sodium azide), and incubated for 20 min on
ice with the indicated Ab, which were either biotinylated or directly
conjugated with FITC (PharMingen, La Jolla, CA). Cells incubated with
biotinylated Ab were then washed and incubated on ice with
streptavidin-conjugated FITC (PharMingen) for an additional 20 min. For
tetramer binding studies, CTL were incubated on ice with PE-conjugated
KdHA tetramer and FITC-conjugated CD8 for 1
h. Samples were then washed twice and immediately analyzed. Tetramers
were produced as previously described (40), except that
KdHA peptide was incorporated. For intracellular
IFN-
expression studies, resting CTL lines and clones were
restimulated for 6 h with B10.D2 fibroblasts pulsed with varying
concentrations of HA peptide in the presence of brefeldin A. Following
incubation, CTL were washed and incubated with CD8 conjugated with
Cychrome (PharMingen) for 20 min. Samples were then permeabilized and
incubated with Ab specific for IFN- or an isotype control Ab, each
conjugated with FITC. Buffers and procedures were supplied in the
Golgi-Plug kit (PharMingen). Following this procedure, CTL were fixed
with 1% paraformaldehyde before flow cytometric analysis. All cells
were analyzed with either a FACScan or FACScalibur instrument, and
postcytometric analysis was performed using Macintosh CellQuest
software (all from Becton Dickinson, Mountain View, CA).
Immunohistochemistry
Pancreata were excised, fixed in 10% (v/v) formalin solution (Sigma), and processed for paraffin embedding. Paraffin-embedded tissue was cut using a microtome, and sections were placed onto saline-coated SuperFrost slides for processing (Fisher Scientific, Pittsburgh, PA). Tissue sections were deparaffinized in xylene and gradually rehydrated in graded aqueous/alcohol solutions, beginning with 100% ethanol and ending in distilled water. Nonspecific binding sites were blocked by incubation with 10% (v/v) goat serum in PBS, followed by incubation for 1 h with Ab specific for murine insulin (Dako, Carpinteria, CA). After washing for 10 min in PBS, sections were incubated with a secondary biotinylated F(ab')2 (Vector, Burlingame, CA) and developed with streptavidin-conjugated HRP using diaminobenzidine as a chromagen (Jackson ImmunoResearch Laboratories, Avondale, PA). Serial sections of paraffin-embedded tissue were also stained with eosin, and all slides were counterstained with Mayer’s hematoxylin (both reagents from Sigma).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The InsHA mice are profoundly tolerant to HA expressed on
pancreatic ß islet cells, and do not develop diabetes after infection
with influenza virus (15). However, low, but detectable,
KdHA-specific CTL activity can be generated from
spleens of InsHA mice previously immunized with influenza, provided the
APCs are pulsed with a high concentration of KdHA
peptide (18). To characterize the CTL that were
responsible for the low levels of lytic activity and to compare them
with KdHA-specific CTL from a nontolerant
repertoire, KdHA-specific CTL were derived from
conventional D2 mice as well as InsHA mice that were homozygous
(InsHA+/+) or hemizygous
(InsHA+/-) for the HA transgene. The CTL from
each of these mice were assessed for lytic activity against target
cells pulsed with various amounts of KdHA
peptide. As reported previously, these results indicated that the
consequence of HA expression in the periphery by InsHA mice was an
HA-specific CTL population of decreased magnitude and avidity compared
with KdHA-specific CTL derived from mice that are
not tolerant of HA (Fig. 1
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(18).
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Considering that the magnitude of the
KdHA-specific CTL response from InsHA mice was
considerably weaker than that observed in nontolerant D2 animals, it
was possible that tolerance could be explained on the basis of this
quantitative difference in the CTL response to islet-expressed HA. If
this were indeed the case, then it would be anticipated that in vitro
expansion of HA-specific CTL from InsHA mice and subsequent adoptive
transfer into InsHA recipients could result in diabetes. To determine
whether tolerance reflected such a quantitative difference in the T
lymphocyte repertoire, HA-specific CTL from previously immunized D2,
InsHA+/- and InsHA+/+ mice
were restimulated and expanded in vitro with APCs pulsed with
KdHA peptide to enrich for HA-specific
CD8+ T lymphocytes. Ten days after stimulation,
3 x 106 total lymphocytes were transferred
into irradiated InsHA+/+ recipients. Fig. 2 focuses on two recipients from each
group. Seven to 14 days following transfer, one of two
InsHA+/+ mice receiving D2-derived CTL became
hyperglycemic (Fig. 2A). Blood glucose levels in all
recipients of InsHA-derived CTL remained euglycemic. Twenty-one days
later, surviving mice were boosted with influenza virus to determine
whether in vivo stimulation would induce autoreactivity by the
transferred InsHA-derived KdHA-specific CTL.
Following such immunization, the other recipient of D2 HA-specific CTL
became hyperglycemic, but recipients of InsHA-derived CTL remained
euglycemic. On day 35, animals were killed, and pancreata were
extracted. Immunohistological examination of pancreata revealed
profound insulitis in the recipients of D2-derived CTL (Fig. 2, B (arrows) and E). When stained with
anti-insulin Ab, islets did not display an intact morphology, and
only islet remnants were consistently detected. In contrast, analyses
of pancreatic tissue from InsHA+/+ recipients of
InsHA+/--derived CTL revealed that only 
20%
of the islets exhibited insulitis, while 50% exhibited peri-insulitis
(Fig. 2, C (arrows) and E). Sections of
pancreatic tissue from InsHA+/+ mice that had
received InsHA+/+-derived CTL displayed even less
infiltration. No insulitis was detected, while peri-insulitis was noted
in 52% of the islets assessed (Fig. 2D, arrows). These
results were similar to those observed in
InsHA+/+ mice that were immunized with PR8 but
did not receive CTL (Fig. 2E). These results demonstrated
that augmenting the number of low avidity, HA-specific CTL within the
periphery of InsHA+/+ mice resulted in
Ag-specific accumulation of lymphocytes around the pancreatic islets.
However, this did not result in either massive destruction of the
islets or diabetes.
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Fas/FasL-mediated lysis has been found to be an important
potentiator of islet destruction (41, 42). In addition,
some CTL specific for self are unable to lyse targets via the perforin
pathway, but are proficient in Fas/FasL-mediated lysis (43, 44). Therefore, it was possible that low avidity
KdHA-specific CTL were not diabetogenic because of an
inability to mediate cytotoxicity through the Fas/FasL pathway. To
address this issue, KdHA-specific
InsHA+/+-derived and D2-derived CTL were compared for
their lytic ability against Fas+ and Fas-
targets in the presence of EGTA, which inhibits perforin-mediated
lysis. In the absence of EGTA, both CTL populations readily lysed HA
peptide-pulsed Fas+ and Fas- targets at levels
significantly higher than those for nonpeptide-pulsed targets, as
expected (Fig. 3, A and
C). However, in the presence of EGTA, InsHA-derived CTL
were proficient in mediating Fas/FasL lysis and were capable of lysing
targets at slightly higher levels than those observed with D2 CTL (Fig. 3B). These results suggested that the inability of InsHA
CTL to cause diabetes was not due to a deficiency in the ability to
mediate lysis through the Fas/FasL pathway.
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Experiments from other investigators have suggested that repeated,
chronic exposure to Ag in the periphery may result in lymphocytes that
are functionally deficient or anergic due to decreased intracellular
activation following stimulation through the TCR complex
(28, 29, 30, 31). To determine whether a defect in activation
through the TCR complex accounted for the apparent lower avidity of
KdHA-specific CTL derived from InsHA mice, target
cell lysis was made Ag independent by direct triggering of lytic
activity through CD3 (45). Fc
receptor+ P815 cells were coated with 2C11 Ab
(specific for CD3
) before the addition of effector CTL. As
demonstrated in Fig. 4, InsHA and D2 CTL
populations were comparable in the ability to lyse P815 targets coated
with varying amounts of 2C11 Ab. Thus, when the contribution of TCR
affinity for KdHA was bypassed, InsHA CTL and D2
CTL were capable of equivalent levels of lytic activity.
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Other investigators have demonstrated that T lymphocytes from TCR
transgenic mice specific for self can decrease their avidity and
survive negative selection in the thymus or periphery by cell surface
down-regulation of TCR or CD8 coreceptor (8, 20, 28, 46).
To address whether this mechanism accounted for the lower avidity of
KdHA-specific CTL derived from InsHA mice, cell
surface expression of TCR and CD8 was assessed by flow cytometry. When
KdHA-specific D2-derived and
InsHA+/+-derived CTL were compared in this
fashion, no difference in cell surface expression of either molecule
was observed (Fig. 5). In addition to TCR
and CD8, other cell surface markers have been shown to contribute to
the overall avidity of TCR/MHC-peptide interactions (47 ;
reviewed in Ref. 48) and cytolytic capability
(49). Flow cytometric analysis was performed to determine
whether KdHA-specific CTL derived from InsHA and
D2 mice differed with respect to various cell surface molecules that
are involved in adhesion or indicate activation. These studies revealed
that the KdHA-specific CTL from both
InsHA+/+ and D2 mice expressed an identical
activation phenotype: CD44high and
CD62Llow, with similar levels of CD25 (Fig. 5). In addition, both populations expressed equal levels of CD11
(LFA-1) and CD49d (VLA-4), molecules involved in cell-cell adhesion and
homing. Extended studies that assessed cell surface expression levels
of other proteins also showed no differences between
InsHA+/+-derived and D2-derived CTL populations
(CD8ß, CD122ß, and CD28; data not shown). Taken together, all
experiments described to date indicated that, compared with high
avidity CTL, the inherently low avidity of InsHA CTL could not be
attributed to deficiencies in effector function or differential
expression of key cell surface glycoproteins.
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Findings from other laboratories have estimated that 7–29% of
peripheral T lymphocytes express two TCR -chains (23, 50), and this mechanism is one way for potentially autoreactive
CTL to exist in the periphery and remain tolerant to self (22, 24). To examine the possibility that
KdHA-specific CTL in InsHA mice were using this
mechanism to down-regulate avidity to HA, it was necessary to perform
analysis of InsHA CTL at the single cell level. To this end, CTL lines
that retained cytolytic activity over several passages were cloned by
limiting dilution. To analyze expression of the TCR locus, purified
poly(A)+ RNA was isolated from each clone and
subjected to RT-PCR, using 5' amplimers specific for individual V
regions and a 3' amplimer specific for the C region in a procedure
originally described by Zisman et al. (36).
Poly(A)+ RNA isolated from three
KdHA-specific D2-derived CTL clones, including
the previously described clone 4 (51), were also used in
these experiments for comparative purposes. Sequencing of the amplified
cDNAs revealed that two KdHA-specific
InsHA-derived CTL clones (InsHA+/- clone 9.1 and
InsHA+/+ 10) had two transcriptionally active
TCR alleles capable of encoding complete TCR -chains (Table I). All D2 clones examined and the
remaining seven InsHA clones were actively transcribing only one
productively rearranged allele. Although two clones
(InsHA+/+ clones 9 and 12) were also found to
transcribe both TCR alleles, sequencing analysis of the cDNAs
revealed that one of the transcripts from each clone contained a
premature stop codon within the CDR3 region (data not shown). These
observations indicated that expression of two TCR -chains among
InsHA-derived KdHA-specific CTL clones may be no
more frequent than what is found in the general T lymphocyte
repertoire.
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To determine whether the KdHA-specific CTL
clones reflected the same affinity disparities observed for the
different starting populations, the avidity of CTL clones was assessed
by analysis of lytic activity against targets pulsed with varying
concentrations of KdHA peptide. The results
depicted in Fig. 6 showed that D2-derived
CTL were observed to have the highest avidity, as depicted by
representative clone 6 (Fig. 6A). Overall, InsHA CTL clones
were of significantly lower avidity, with
InsHA+/- clones 1 and 8 exhibiting somewhat
higher lytic activity than the InsHA+/+ clones 9,
10, and 12.
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(52). Therefore, it was of interest to determine whether
the avidity differences reflected in lytic activity by the various CTL
clones also pertained to production of this proinflammatory cytokine.
Seven days poststimulation, CTL clones were restimulated for 6 h
with target cells pulsed with the indicated amount of peptide. At the
end of the incubation, CTL were stained with anti-CD8 before
intracellular staining with anti-IFN-. Using this approach, it
was observed that D2 clone 6 expressed the highest amounts of IFN-,
and this expression was sustained even when stimulated with target
cells pulsed with low amounts of peptide (Fig. 6
B). At lower
peptide concentrations, InsHA+/- clone 1
expressed significantly less IFN- than D2 clone 6, but expression
was higher than that observed with InsHA+/+ clone
12. Taken together, the results in Fig. 6 indicated that the overall
avidity differences between T lymphocytes from tolerant and nontolerant
mice were reflected in cytokine production and triggering of lytic
activity. Analysis of TCR affinity by binding to Kd-HA tetramers
The results presented above strongly suggested that the affinity
of the TCR receptor for the KdHA complex may well
be the only significant difference between diabetogenic CTL from
nontolerant mice and most nondiabetogenic CTL from tolerant mice. To
determine whether an affinity difference was reflected in the ability
of these CTL clones to bind the KdHA complex,
selected D2 and InsHA clones were compared with respect to their
ability to bind KdHA tetramers. An
HLA-A2/Kb-restricted CTL clone 12, specific for
murine p53261–269 was used as a negative
control. The results depicted in Fig. 7
showed significant variation in tetramer binding by CTL clones, and
that this variability correlated with avidity for HA. The D2-derived
CTL clones bound significantly more tetramer than any InsHA-derived CTL
clone. In addition, InsHA+/- clones 1 and 8
bound tetramer less efficiently than D2 clones 6 and 17, but more
efficiently than InsHA+/+ clones 9, 10, and 12
(Fig. 7). As anticipated, the weakest binding was exhibited by the
noncognate HLA-A2/Kb-restricted CTL clone. TCR
expression levels of each clone were also assessed, because TCR
expression has a direct correlation with tetramer binding
(53). Analysis of the data revealed that high TCR
expression by InsHA CTL clones did not necessarily correlate with
higher tetramer binding (Fig. 8). In
fact, InsHA+/- clone 8 and
InsHA+/+ clone 9 consistently expressed higher
levels of TCR than D2 CTL clones 6 and 17, but demonstrated
significantly less binding of KdHA tetramers.
These data indicate that the overall low avidity of
KdHA-specific InsHA-derived CTL observed in
functional studies can be correlated with possession of a TCR with
lower affinity, as measured by the ability to specifically bind
KdHA tetramers.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (22, 24, 56), reduction in levels of TCR or
CD8 (8, 20, 21, 28), or anergy (28, 30, 31).
However, to date, no study has been conducted that assesses whether
these mechanisms actually pertain to residual anti-self T
lymphocytes that persist within a conventional repertoire. In this
study our goal was to determine the molecular basis for the low avidity
displayed by HA-specific CD8+ T lymphocytes that
persist within the repertoire of functionally tolerant InsHA mice. In
addition, these studies compared the functional capacities of high and
low avidity CD8+ T lymphocytes with respect to
their pathogenicity in a diabetes model. Previously in this same model, it was demonstrated that HA-specific CTL in InsHA mice were unable to initiate diabetes (15, 18). This could have been due to either a quantitative or a qualitative deficiency in the HA-specific CD8+ T lymphocyte repertoire. The KdHA-specific InsHA-derived CTL were of lower avidity than D2-derived CTL, and one consequence of low avidity is likely to be less in vivo clonal expansion in response to Ag (57, 58). We expanded the numbers of low avidity KdHA-specific CTL within the repertoire of InsHA mice by transferring CTL that were previously activated in vitro with high concentrations of peptide. Although InsHA-derived CTL failed to induce diabetes, this was not due to a deficiency in the ability to home to the pancreas. KdHA-specific, InsHA-derived CTL were capable of detecting HA on ß islet cells, as evidenced by the occurrence of peri-insulitis. However, there was little detectable ß islet cell destruction, and no occurrence of diabetes in these recipients, even after immunization with influenza virus. Therefore, the autoimmune potential of InsHA-derived KdHA-specific CTL was not increased by simply augmenting their numbers. Furthermore, the observed qualitative difference in avidity between InsHA and D2 CTL in vitro predicted the destructive capacity of InsHA CTL in vivo. This observation reflects earlier reports that avidity differences between CTL lines in vitro correlated with the success of viral clearance in vivo (33, 59).
The degree of HA-specific lymphocyte infiltration in the pancreas was much greater when KdHA-specific CTL derived from InsHA+/- mice were transferred than with CTL derived from InsHA+/+ mice. This probably reflects the in vitro differences in cytolytic activity observed for these CTL, both as populations and as clones, and demonstrates the principle that expression of greater amounts of peripheral Ag results in a more profoundly tolerized repertoire, as reflected in the lower avidity of CTL from InsHA+/+ mice. This correlation has been previously observed in the CD4+ T lymphocyte repertoire of transgenic mice expressing either beef insulin (60) or varying levels of hen egg white lysozyme in vivo (61).
Extensive analyses were performed to determine the molecular basis for both the low avidity and the lack of diabetogenic capacity of the KdHA-specific CTL derived from InsHA mice. We first assessed how well InsHA-derived KdHA-specific CTL performed in mediating lysis through the Fas/FasL pathway, as other investigators have observed that destruction of Fas+ ß cells solely through this pathway can initiate autoimmune diabetes in mouse models (41, 62). However, InsHA-derived KdHA-specific CTL were proficient in lysing targets when the perforin pathway was inhibited. We next asked whether InsHA-derived CTL possessed an inherent deficiency in the ability to mediate activation through CD3, if Ag recognition was bypassed. There is precedence for low avidity CTL to be deficient in intracellular signaling. Activation of CTL isolated with high peptide concentrations has been observed to require higher concentrations of anti-CD3 Ab than higher avidity clones generated by isolation with 1000-fold less peptide (33). In another report intracellular signaling deficiencies were detected in CD8+ T lymphocytes after exposure to multiple in vivo peptide injections (31). However, both high and low avidity KdHA-specific CTL responded equivalently to anti-CD3, suggesting that comparable stimulation of signal transduction through the TCR could occur when the specificity and affinity of the TCR were bypassed.
In several models tolerance has been accomplished through
down-regulation of TCR and CD8. High avidity T lymphocytes isolated
from TCR transgenic mice appear to readily down-regulate expression of
the TCR or CD8 coreceptor after exposure to Ag in vivo (8, 20, 46, 63). T lymphocytes with decreased expression of TCR or CD8
cannot properly respond to antigenic stimulation and in some instances
appear anergic. This mechanism was not employed by HA-specific T
lymphocytes from InsHA mice, as differences in expression of TCR, CD8,
as well as other cell surface glycoproteins involved in T lymphocyte
activation were not observed (Fig. 5). However, it must be considered
that our study only involved CD8+ T lymphocytes
that successfully proliferated, both in vivo and in vitro, in response
to HA. CD8+ T lymphocytes that had down-regulated
molecules necessary for activation and proliferation would not
necessarily have been detected in these studies. Additionally, it is
possible that some of the differences between the current study and the
results obtained using TCR transgenics may have to do with homeostatic
mechanisms that may dictate different outcomes for survival of
tolerized T lymphocytes within a conventional repertoire vs a TCR
transgenic repertoire. In a conventional repertoire, T lymphocytes that
down-regulate TCR or CD8 may be quickly eliminated, while T lymphocytes
originating from a transgenic repertoire may persist in vivo
(64).
Although no evidence for down-regulation of CD8 or TCR was obtained by
comparing populations of HA-specific CTL from D2 and InsHA mice,
analysis at the clonal level did reveal that heterogeneity in TCR
expression can occur. InsHA+/- clone 8
demonstrated higher levels of TCR expression and tetramer binding than
InsHA+/+ clone 12. As both clones were found to
express a single TCR, it is possible that the avidity differences
observed may at least in part be attributed to differences in TCR
expression levels rather than different affinities for
KdHA (53). Also, the possibility
must be considered that the relatively low avidity of
InsHA+/+ clone 10, which transcribes two TCR
-chains and therefore may express two TCR heterodimers, may also
reflect low level expression of the HA-specific TCR. Previous studies
have shown that expression of a second TCR -chain can modulate the
response of a T lymphocyte by decreasing the overall avidity for
cognate Ag (24). Studies assessing TCR usage among
KdHA-specific CTL clones from InsHA mice
indicated that seven of nine clones expressed a single productively
rearranged TCR -chain. The in vitro selection method used in this
study to isolate CTL clones required lytic activity against targets
presenting the KdHA epitope. This stringent
requirement may have precluded isolation of more CTL that possessed two
functional TCR rearrangements. However, the finding that two of nine
InsHA-derived clones (22%) expressed two functional TCR -chains
falls in the range of estimations of T lymphocytes within a
conventional murine repertoire that express two TCR -chains
(7–29%) (23, 50). This would not support the hypothesis
that most T cells escape tolerance through expression of a second TCR
heterodimer. Nevertheless, it would be of interest to determine whether
clones such as InsHA+/+ clone 10 can be
manipulated in vitro or in vivo to express more or less of the
KdHA-specific TCR heterodimer, and whether this,
in turn, alters the diabetogenic capacity of the clone.
Taken together with the other results in this study, the fact that most
of the InsHA-derived CTL clones exhibited expression of a single TCR
-chain strongly suggested that for most
KdHA-specific CTL, differences in avidity and
diabetogenic capacity were the result of differences in TCR affinity.
Peptide-MHC tetramers have been used to correlate tetramer binding with
class II MHC-restricted TCR affinity (53), track the
expansion of high affinity Ag-specific T lymphocytes during an immune
response (40, 65), and compare the affinities of primary
and secondary T lymphocytes (57). Moreover, FACS
purification of CTL with high tetramer binding efficiency from a
heterogeneous population resulted in CTL clones displaying enhanced
avidity (66). It was anticipated that there would be a
direct correlation between the level of binding of
KdHA tetramers and CTL avidity. This was indeed
the case, as binding of tetramers by the CTL clones correlated with the
avidity observed in functional studies: D2 >
InsHA+/- > InsHA+/+.
Higher expression of TCR did not necessarily increase tetramer
reactivity. For example, InsHA+/+ clone 9
expressed TCR at levels higher than those of any other CTL clone while
simultaneously displaying the lowest tetramer binding efficiency.
Similarly, CD8 expression was somewhat variable from clone to clone,
but did not correlate with tetramer reactivity or whether the clone was
of D2 or InsHA origin (data not shown). The high binding of D2 clones
and the lower binding of InsHA clones with tetramer further support the
conclusion that the low avidity of InsHA CTL can be explained on the
basis of TCR affinity. Based on current understanding of the
correlation between T lymphocyte triggering and TCR off-rate, it is
likely these differences in tetramer binding reflect differences in the
rate of dissociation of MHC-peptide from the TCR.
These studies have evaluated some of the phenotypic and functional
properties that distinguish T lymphocytes from tolerant or nontolerant
situation. Our results argue that most of the
KdHA-specific T lymphocytes in InsHA mice are not
intrinsically different from other T lymphocytes; they simply express
TCRs that are less avid in binding of KdHA. This
supports the view that tolerance to peripherally expressed HA is
maintained by deletion of high affinity HA-specific lymphocytes, and
that low affinity lymphocytes remain, most likely by virtue of their
failure to be activated and deleted by the peripheral Ag (16, 17). As a result, HA-specific T lymphocytes require
significantly more KdHA peptide to be triggered
for effector functions such as secretion of IFN- and cytolytic
activity. Under normal circumstances, this requirement is sufficient to
preclude autoimmunity. However, there may be situations in which these
lymphocytes become capable of autoimmune destruction, such as
activation within a proinflammatory environment that has been created
due to genetic or environmental factors (67, 68, 69) or
triggering by an Ag that is recognized with greater affinity than a
self (HA) peptide (70, 71). Future experiments will
investigate these possibilities.
|
Footnotes |
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2 Address correspondence and reprint requests to Dr. Linda A. Sherman, IMM-15, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:
3 Abbreviations used in this paper: HA, influenza virus hemagglutinin; D2, B10.D2 mice; InsHA mice, D2 mice expressing the HA transgene under control of the rat insulin promoter; FasL, Fas ligand.
4 J. Hernandez and L. Sherman. Submitted for publication.
Received for publication August 18, 1999. Accepted for publication October 12, 1999.
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References |
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300 mg/dl. Twenty-one days
posttransfer, surviving recipients were immunized with PR8. Upon
termination of the experiment (day 35), pancreata of recipients were
processed, stained for insulin expression, and scored for islet damage.
Arrows designate lymphocytes, and n designates ß islet
cell nuclei. B, Adoptive transfer of a D2 CTL population
(x200). Note the presence of a mononuclear cell infiltrate throughout
the islet, leaving very few insulin-positive ß cells.
C, Adoptive transfer of an InsHA+/- CTL
population (x200). ß islet cell clusters are intact with normal
morphology and express uniform levels of insulin, but peri-insulitis is
evident. Note the peripheral localization of mononuclear lymphocytes.
D, Adoptive transfer of an InsHA+/+ CTL
population (x100). As in B, peri-insulitis is evident
in these mice, although to a much lesser degree. E,
Islet numbers from each sample in this experiment were quantitated and
scored for insulitis. The numbers in parentheses indicate the number of
islets detected from each experimental group. For negative controls,
samples were also taken from recipients that did not receive CTL and
were only irradiated or were irradiated and immunized with PR8. The
results in this experiment reflect observations made with three
additional mice per transferred CTL population. A total of four of five
D2 CTL recipients became hyperglycemic, and all recipients of
InsHA+/- and InsHA+/+ CTL remained
euglycemic.
