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The Journal of Immunology, 2000, 164: 191-200.
Copyright © 2000 by The American Association of Immunologists

Characterization of CD8+ T Lymphocytes That Persist After Peripheral Tolerance to a Self Antigen Expressed in the Pancreas1

C. Thomas Nugent{dagger}, David J. Morgan{dagger}, Judith A. Biggs{dagger}, Alice Ko{dagger}, Ingrid M. Pilip*, Eric G. Pamer* and Linda A. Sherman2,{dagger}

* Sections of Infectious Diseases and Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and {dagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As a result of expression of the influenza hemagglutinin (HA) in the pancreatic islets, the repertoire of HA-specific CD8+ T lymphocytes in InsHA transgenic mice (D2 mice expressing the HA transgene under control of the rat insulin promoter) is comprised of cells that are less responsive to cognate Ag than are HA-specific CD8+ T lymphocytes from conventional mice. Previous studies of tolerance induction involving TCR transgenic T lymphocytes suggested that a variety of different mechanisms can reduce avidity for Ag, including altered cell surface expression of molecules involved in Ag recognition and a deficiency in signaling through the TCR complex. To determine which, if any, of these mechanisms pertain to CD8+ T lymphocytes within a conventional repertoire, HA-specific CD8+ T lymphocytes from B10.D2 mice and B10.D2 InsHA transgenic mice were compared with respect to expression of cell surface molecules, TCR gene utilization, binding of tetrameric KdHA complexes, lytic mechanisms, and diabetogenic potential. No evidence was found for reduced expression of TCR or CD8 by InsHA-derived CTL, nor was there evidence for a defect in triggering lytic activity. However, avidity differences between CD8+ clones correlated with their ability to bind KdHA tetramers. These results argue that most of the KdHA-specific T lymphocytes in InsHA mice are not intrinsically different from KdHA-specific T lymphocytes isolated from conventional animals. They simply express TCRs that are less avid in their binding to KdHA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Two conflicting goals must be met during selection of the CD8+ TCR repertoire. First, to optimize the potential value of the CD8+ T lymphocyte to the host, only those thymocytes that express receptors that recognize self class I MHC-peptide complexes (self) are selected for further maturation (1, 2). Recognition of self is also required for the continued survival of the T lymphocyte in the periphery (3). Second, to avoid autoimmunity, the avidity for self must be below the threshold necessary to induce T lymphocyte activation. All CD8+ T lymphocytes that exceed this threshold are deleted. Deletion begins in the thymus and can occur in the periphery, where T lymphocytes expressing TCRs specific for peripherally expressed Ags are eliminated if they encounter a professional APC expressing cognate Ag (4, 5, 6, 7, 8, 9).

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 {alpha}-chain (22, 23, 24, 25, 26). This results in decreased expression of the potentially autoimmune TCR {alpha}/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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

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{alpha} transcript expression, we used amplimers and procedures previously described by Zisman et al. (36). Briefly, 5' amplimers specific for individual V{alpha} genes were used in conjunction with a 3' amplimer specific for C{alpha} 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{alpha} 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{alpha}- and J{alpha}-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-{gamma} 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-{gamma} 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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of KdHA-specific CTL from D2 and InsHA mice

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. 1Go) (18).



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  		FIGURE 1. Comparison of HA-specific CTL populations from D2 and InsHA mice. Single-cell suspensions of spleens from previously immunized mice were restimulated twice with irradiated APC pulsed with HA peptide for 7 days. Four days following the second passage, CTL were harvested and incubated with peptide-pulsed target cells in a standard 4-h chromium-release assay. The results in this experiment reflect observations from three additional experiments.

 
Comparison of the ability of D2 and InsHA CTL to initiate diabetes in InsHA recipients

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. 2Go 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. 2GoA). 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. 2Go, 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. 2Go, 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. 2GoD, arrows). These results were similar to those observed in InsHA+/+ mice that were immunized with PR8 but did not receive CTL (Fig. 2GoE). 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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  		FIGURE 2. Adoptive transfer of CTL into InsHA+/+ recipients. Single-cell suspensions from spleens of previously immunized D2, InsHA+/-, or InsHA+/+ mice were restimulated once with irradiated APC and cultured for 10 days as described in Materials and Methods. CTL were harvested, washed, and found by flow cytometric analysis to be 18–25% CD8+ T lymphocytes. Three million cells were injected i.v. into irradiated InsHA+/+ recipients. A, Animals were bled retro-orbitally every 7 days to monitor blood sugar levels, using an Accu-Chek III monitor (Roche Molecular Biochemical, Indianapolis, IN). Mice were considered hyperglycemic and were sacrificed when blood glucose was >=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.

 
Comparison of the lytic mechanism employed by HA-specific CTL from tolerant and nontolerant mice

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. 3Go, 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. 3GoB). 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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  		FIGURE 3. Analysis of the Fas/FasL lytic pathway usage by D2 and InsHA CTL. Single-cell suspensions from spleens of previously immunized D2 or InsHA+/+ mice were restimulated twice with irradiated APC as described in Materials and Methods. Five days after the second round of stimulation, activated CTL were incubated with peptide-pulsed or mock-pulsed target cells that were either positive (A and B) or negative (C and D) for Fas expression in a 5-h chromium release assay. To independently assess Fas/FasL-specific lysis, 4 mM EGTA was added to a duplicate assay to inhibit the activity of perforin (B and D).

 
Both InsHA and D2 CTL are comparable in their ability to signal through CD3

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{epsilon}) before the addition of effector CTL. As demonstrated in Fig. 4Go, 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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  		FIGURE 4. Analysis of redirected, Ag-independent, CD3-mediated lysis. Single-cell suspensions from spleens of previously immunized D2 or InsHA+/+ mice were restimulated twice with irradiated APC as described in Materials and Methods. Five days after the second round of stimulation, activated CTL were cocultured for 4 h with chromium-labeled P815 target cells previously incubated with the indicated concentrations of 2C11 Ab, which is specific for CD3{epsilon}. The percent specific lysis of P815 cells not coated with 2C11 Ab was <10% for D2 CTL and <5% for InsHA CTL.

 
Comparison of cell surface expression of molecules involved in CD8+ T lymphocyte activation

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. 5Go). 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{alpha} (Fig. 5Go). 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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  		FIGURE 5. Flow cytometric analysis of cell surface markers. Single-cell suspensions from spleens of previously immunized D2 or InsHA+/+ mice were restimulated twice with irradiated APC as described in Materials and Methods. Four days after the second round of stimulation, HA-specific CTL from InsHA or D2 mice were incubated for 20 min with the indicated FITC-conjugated Ab (PharMingen). Isotype Ab directly conjugated with FITC were used as negative controls. Analysis of samples was conducted using a FACScan and CellQuest software (Becton Dickinson).

 
Isolation and characterization of KdHA-specific InsHA and D2 CTL clones

Findings from other laboratories have estimated that 7–29% of peripheral T lymphocytes express two TCR {alpha}-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{alpha} locus, purified poly(A)+ RNA was isolated from each clone and subjected to RT-PCR, using 5' amplimers specific for individual V{alpha} regions and a 3' amplimer specific for the C{alpha} 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{alpha} alleles capable of encoding complete TCR {alpha}-chains (Table IGo). 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{alpha} 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 {alpha}-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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  		Table I. TCR-{alpha} expression by CTL clones1

 
Functional characterization of KdHA-specific CTL clones

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. 6Go showed that D2-derived CTL were observed to have the highest avidity, as depicted by representative clone 6 (Fig. 6GoA). 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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  		FIGURE 6. Clone avidity analyzed by HA-specific lysis and IFN-{gamma} production. A, Activated CTL clones (stimulated 4 days previously) were coincubated with chromium-labeled target cells pulsed with the indicated amount of KdHA peptide in a standard 4-h chromium release assay. B, Three days later, additional CTL were purified by Ficoll gradient and restimulated in the presence of target cells pulsed with the indicated amount of KdHA peptide for 6 h in the presence of brefeldin. Samples were then analyzed for cell surface expression of CD8 (using a cychrome-labeled anti-CD8 Ab, PharMingen) and intracellular levels of IFN-{gamma} (using an FITC-labeled Ab specific for IFN-{gamma}, PharMingen). The percentages of CD8+/IFN-{gamma}+ CTL were determined using a two-parameter density plot (CD8-Cyc vs IFN-{gamma}-FITC) generated with CellQuest software (Becton Dickinson).

 
Another effector function of CD8+ T lymphocytes that is critical for ß islet destruction is production of IFN-{gamma} (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-{gamma}. Using this approach, it was observed that D2 clone 6 expressed the highest amounts of IFN-{gamma}, and this expression was sustained even when stimulated with target cells pulsed with low amounts of peptide (Fig. 6GoB). At lower peptide concentrations, InsHA+/- clone 1 expressed significantly less IFN-{gamma} than D2 clone 6, but expression was higher than that observed with InsHA+/+ clone 12. Taken together, the results in Fig. 6Go 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. 7Go 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. 7Go). 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. 8Go). 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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  		FIGURE 7. KdHA tetramer binding and lytic activity of HA-specific CTL clones. Activated CTL clones were harvested, washed, and purified by Ficoll gradient before resuspension in FACS buffer. Five hundred thousand lymphocytes per clone were then incubated with FITC-labeled anti-CD8 Ab and H57 anti-TCR-PE Ab (PharMingen) or with FITC anti-CD8 Ab and PE KdHA tetramer for 1 h. Samples were washed twice and analyzed.

 


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  		FIGURE 8. Scatterplot analysis of TCR expression and tetramer reactivity. The mean fluorescence for TCR and the tetramer reactivity of each clone were determined from the flow cytometric data generated in Fig. 7Go and were graphed in scatterplot fashion to relatively compare TCR Ab and KdHA tetramer binding by CTL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Self-reactive lymphocytes are a normal component of the immune systems of healthy organisms (54, 55), and the intriguing possibility of regulating these lymphocytes would impact the control of autoimmunity and promote cancer immunotherapy. Most research characterizing self-reactive lymphocytes has focused on how the various mechanisms of tolerance induction affect the fate of T lymphocytes that demonstrate high affinity for self. Such studies have also elucidated mechanisms by which T lymphocytes expressing high affinity TCRs may become reduced in their avidity such that spontaneous autoimmunity does not occur. Examples of these mechanisms include expression of a second TCR {alpha}-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. 5Go). 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 {alpha}-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 {alpha}-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 {alpha}-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{alpha} rearrangements. However, the finding that two of nine InsHA-derived clones (22%) expressed two functional TCR {alpha}-chains falls in the range of estimations of T lymphocytes within a conventional murine repertoire that express two TCR {alpha}-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 {alpha}-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-{gamma} 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
 
1 This work was supported by National Institutes of Health Grants AI39664/JDF995010 and DK/CA50824 (to L.A.S.) and Juvenile Diabetes Foundation Fellowship 398335 (to C.T.N.). Back

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: Back

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. Back

4 J. Hernandez and L. Sherman. Submitted for publication. Back

Received for publication August 18, 1999. Accepted for publication October 12, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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