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Identical Cell-Specific CD8⁺ T Cell Clonotypes Typically Reside in Both Peripheral Blood Lymphocyte and Pancreatic Islets¹

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

	Abstract

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A major issue regarding T cell responses in autoimmunity is how the repertoire compares between the periphery and target organ. In type 1 diabetes, the status of at-risk or diabetic individuals can be monitored by measuring cell-specific T cells isolated from PBL, but whether these T cells accurately reflect the repertoire residing in the pancreatic islets is unclear. The TCR repertoire of disease-relevant, tetramer-sorted CD8⁺ T cells was examined at the single-cell level in PBL, pancreatic lymph nodes (PLN), and the islets of individual NOD mice. CDR3 and CDR3 sequences demonstrated that the same repertoire of T cells in PBL was detected in the islets and PLN, although the frequency of specific clonotypes varied. Albeit infrequent, clonotypes that were prevalent in the islets but not found in PBL were also detected. cell Ag immunization expanded immunodominant PBL clonotypes present in the islets and PLN. These results show that insight into repertoire profiles of islet-infiltrating T cells can be obtained from PBL.

	Introduction

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A key question in T cell immunology is how the repertoire at one site reflects the repertoire at distinct anatomical locations. This is of considerable importance in both tissue-targeted infections and in organ-restricted autoimmune diseases such as type 1 diabetes (T1D).³ Insight into the nature of an immune response can be gained by knowing whether T cells found in blood represent 1) a broad spectrum of clonotypes some of which are involved in mediating a localized response, or 2) a selected population identical with those effectors residing in the target tissue.

Autoimmune destruction of the insulin-producing cells in T1D is mediated by CD4⁺ and CD8⁺ T cells (1, 2, 3, 4). Several cell autoantigens are targeted by T cells in diabetic patients and the NOD mouse, a model for T1D (1, 2, 3, 4). An H2K^d-restricted peptide derived from islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)_206–214 is recognized by a high frequency of CD8⁺ T cells residing in islet infiltrates of NOD mice (5, 6). Several lines of evidence suggest that IGRP_206–214-specific CD8⁺ T cells play a critical role in T1D. First, the frequency and TCR avidity of IGRP_206–214-specific CD8⁺ T cells increase in the islet infiltrates during disease progression (7, 8). Second, diabetes onset is accelerated in NOD mice that express a transgenic IGRP_206–214-specific TCR (9). Third, depletion of IGRP_206–214-specific CD8⁺ T cells via treatment with the mimetic peptide NRP-A7 protects NOD mice from diabetes (8, 10). Finally, elevated IGRP_206–214-specific CD8⁺ T cells in PBL of prediabetic NOD mice correlates with the progression to overt diabetes (11).

Tracking changes in cell-specific T cells may provide an accurate readout for disease progression in at-risk or diabetic patients. cell-specific T cells have been detected in PBL of patients and rodent models using tetramer technology or sensitive ELISPOT assays (12, 13, 14). Nevertheless, whether autoreactive T cell clones detected in PBL are identical with those found in the islets and/or at sites of T cell activation such as the draining pancreatic lymph nodes (PLN) has yet to be ascertained (15). We used a single cell-based PCR sequencing method to determine the - and -TCR repertoire of IGRP_206–214-specific CD8⁺ T cells (16, 17) in PBL, the PLN, and islet infiltrates of unmanipulated or immunized NOD mice.

	Materials and Methods

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Mice

NOD/LtJ mice were bred and housed under specific pathogen-free conditions. Each mouse used in this study was derived from an independent litter. Animal protocols were approved by the University of North Carolina Institutional Animal Care Committee.

Tetramers, Abs, and FACS

H2K^d monomers (18) were complexed with NRP-V7 (KYNKANVFL) or NP_147–155 (TYQRTRALV). Tetramers were assembled by conjugating H2K^d monomers with streptavidin-PE (Molecular Probes). Anti-CD3-FITC and anti-CD8-allophycocyanin mAbs were purchased from eBioscience.

T cells were costained with tetramers and Abs in PBS containing 3% FBS, 10 mM HEPES, and 1 mM EDTA for 1 h on ice. FACS data were acquired on a FACSCalibur (BD Biosciences) and analyzed using Summit software (DakoCytomation). For all tetramer analyses, CD8⁺ T cells were gated based on forward and side scatter, and CD3 and CD8 expression. For single-cell analyses, tetramer⁺ CD8⁺ T cells were sorted by a MoFlo high-speed sorter (DakoCytomation) at 1 cell/well into a 96-well PCR plate (USA Scientific), each well containing 4 µl buffer of 0.5x PBS, 10 mM DTT, and 8 U RNaseOUT RNase inhibitor (Invitrogen Life Technologies). Plates were kept frozen at –80°C.

Islet isolation

Islets were purified (17) and cultured overnight in RPMI 1640 containing 10% FBS and 4 ng/ml recombinant murine IL-2 (PeproTech). Lymphocytes infiltrating the islets were collected and cellular debris removed by 70-µm nylon filters before FACS.

Single-cell RT-PCR and TCR repertoire analyses

TCR usage was analyzed by a single-cell RT-PCR protocol (16, 17). TCR -chain analysis was performed using a V17-specific primer in combination with an -chain constant region primer for RT-PCR because all IGRP_206–214-related clonotypes have an invariant V17 gene usage (19, 20). For TCR -chain analysis, a panel of primers specific for all known TCR -chain variable regions in combination with a -chain constant region primer was used. RT-PCR amplicons were used as templates for a second round of PCR amplification using a panel of nested TCR - or -chain-specific primers. Efficiency of RT-PCR for V and V gene segments was 95 and 60–95%, respectively. PCR products were treated with Exonuclease I (NEB Biolabs) and shrimp alkaline phosphatase (Roche) and sequenced by the UNC Sequencing Core Facility. TCR sequence alignments were performed using Sequencher software (Gene Codes).

Generation of Venezuelan equine encephalitis virus replicon particles (VRP)

To generate Ag-expressing, nonreplicating VRP, cDNAs encoding murine IGRP_206–214 (VYLKTNVF), or influenza hemagglutinin (HA)_512–520 (IYSTVASSL)) fused to IgGFc were subcloned into pVR21. RNA was transcribed from plasmids encoding VRP-IGRP or VRP-HA and capsid and envelope glycoprotein using mMESSAGE mMACHINE T7 Kit (Ambion). In vitro-transcribed RNAs were electroporated into baby hamster kidney cells that were cultured for 24 h at 37°C at 5% CO₂. VRP were harvested, concentrated, titered, and stored in PBS plus 1% FCS at –80°C (21). Mice were immunized via footpad with 5 x 10⁵ VRP-IGRP or VRP-HA infectious units in PBS. Ten days after VRP immunization, PBL, PLN, and islets were harvested and tetramer⁺ CD8⁺ T cells were isolated.

Statistical analysis

Statistical analyses were performed using GraphPad Prism (GraphPad). Values of p were calculated using Student’s t test or Fisher’s exact test.

	Results

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IGRP_206–214-specific CD8⁺ TCR repertoire is similar in PBL, PLN, and islets

Because islet-infiltrating T cells are inaccessible in humans, it is critical to know whether peripheral T cell clones are also found in the islets. To address this question, the TCR repertoire of IGRP_206–214-specific CD8⁺ T cells residing in PBL, draining PLN, and islets of recent onset diabetic or euglycemic 20-wk-old NOD female mice was compared. At 20 wk of age, a high frequency of intrainsulitis is detected in the islets of nondiabetic NOD female mice. H2K^d (K^d) tetramers complexed with the high-affinity NRP-V7 mimetic peptide (K^d-V7) were used to identify IGRP_206–214-specific CD8⁺ T cells. Consistent with previous reports, K^d-V7 bound IGRP_206–214-specific CD8⁺ T cells isolated from 8.3 TCR NOD transgenic mice with increased avidity relative to K^d tetramers complexed with native IGRP_206–214 (data not shown) (17). In PBL, PLN, and islets, 1.67 ± 0.74, 0.51 ± 0.27, and 2.21 ± 0.94% of CD8⁺ T cells bound K^d-V7, respectively, whereas staining with control K^d tetramers complexed with an influenza-derived nucleoprotein peptide NP_147–155 (K^d-NP) was minimal (Fig. 1). Single K^d-V7⁺ CD8⁺ T cells were sorted from PBL, PLN, and islets of five individual mice, and TCR -chain usage was determined via single-cell RT-PCR. In agreement with earlier work, K^d-V7⁺ CD8⁺ T cells exclusively used TCR -chain V17 and J42 elements, with the former further divided into three distinct genes, namely V17.4, V17.5, and V17.6 (Table I) (7).

View larger version (30K): [in this window] [in a new window]   FIGURE 1. IGRP206–214-specific CD8+ T cells are detected in PBL, PLN, and islets of NOD mice. A, Representative FACS plots showing Kd-V7+ and (B) control Kd-NP+ CD8+ T cells in PBL (mouse #2), PLN (mouse #3), and islet infiltrates (mouse #4). Events were gated on CD3+ and CD8+ T cells. C, Summary of Kd-V7+ CD8+ cells in PBL, PLN, and islets of five individual female NOD mice 20 wk old. Data represent average tetramer+ CD8+ T cells (±SEM).

View this table: [in this window] [in a new window]   Table I. V17 gene element usage in female NOD mice

V17

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Comparison of TCR CDR3 repertoire of IGRP206–214-specific CD8+ T cells in PBL, islets, and PLN. CDR3 usage of IGRP206–214-specific CD8+ T cells in PBL, islets, and PLN were determined by single-cell RT-PCR in five individual NOD female mice 20 wk old that were diabetic (#2, #4, #5) or euglycemic (#1, #3). The number of CDR3 sequences analyzed in PBL, islets, and PLN, respectively, were as follows: mouse #1 22, 28, 31; mouse #2 27, 36, 34; mouse #3 42, 31, 42; mouse #4 12, 34, 33; mouse #5 11, 19, and 24. *, p 0.05, PBL vs islet; , p 0.01, PBL vs PLN; , p 0.02, islets vs PLN; Fisher’s exact test.

V8.1

J1.3

J2.4

J2.7

View this table: [in this window] [in a new window]   Table II. TCR CDR3 repertoire in single-cell-sorted Kd-V7+ CD8+ T cells in female NOD mice

The T cell repertoire following immunization with IGRP_206–214 was assessed. The goal was to determine whether distinct clonotypes expanded in the periphery were also found in the islets. A possible clinical scenario would be the monitoring of PBL for cell-specific T effectors induced by Ag-specific immunotherapy. Nondiabetic NOD female mice 15 wk of age received a single injection of nonreplicating VRP-IGRP encoding an IGRP_206–214-Ig fusion molecule (22, 23). Preliminary studies indicate that diabetes is prevented in female NOD mice by targeting IGRP_206–214-specific CD8⁺ T cells via Ag-encoding VRP vaccines (R. Tisch, unpublished results). Ten days later, the frequency of K^d-V7⁺ CD8⁺ T cells in PBL was compared with prevaccination levels. As expected, expansion of IGRP_206–214-specific CD8⁺ T cells was readily induced by VRP-IGRP (Fig. 3, A and B). For the five NOD mice, an average increase of 14.1-fold was detected in postvaccination PBL. K^d-V7⁺ CD8⁺ T cells increased from 0.36 ± 0.09 to 5.06 ± 0.52% of CD8⁺ T cells (p < 10^–3, Student’s t test). Importantly, expansion of K^d-V7⁺ CD8⁺ T cells was IGRP_206–214-specific. No significant increase in IGRP_206–214-specific CD8⁺ T cells was detected in PBL from three age-matched NOD mice vaccinated with VRP-HA encoding an HA_512–520-Ig fusion (Fig. 3A). In pre- and post-VRP-HA PBL samples, 0.58 ± 0.14 and 0.83 ± 0.17%, respectively, of CD8⁺ T cells were K^d-V7⁺.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Expansion of IGRP206–214-specific CD8+ T cells in PBL of VRP-IGRP-vaccinated mice. A, Representative FACS plots showing expansion of IGRP206–214-specific CD8+ T cells in PBL of 15-wk-old NOD mice vaccinated with 5 x 105 VRP-IGRP, or VRP-HA via footpad. IGRP206–214-specific CD8+ T cells in PBL were stained with Kd-V7 pre- and 10 days post-VRP vaccination. Events were gated on CD3+ and CD8+ T cells. B, Five NOD mice 15–20 wk old each received a single footpad vaccination of 5 x 105 VRP-IGRP infectious units. Frequencies of Kd-V7+ CD8+ T cells were determined pre- () and postvaccination () in PBL of each mouse. Numbers represent the fold increase of Kd-V7+ CD8+ T cells in each mouse (percentage of Kd-V7+ CD8+ T cells in PBL post- vs prevaccination). V (C), V (D), and J (E) usage were determined by single-cell RT-PCR analysis using Kd-V7+ CD8+ T cells sorted from PBL of individual mice before VRP-IGRP vaccination or from PBL, islets, and PLN 10 days after VRP vaccination. Data represent averaged frequencies of V, V, and J gene families detected from each sample (±SEM).

V17.4

V17.5

V17.6

V8.1

J2.4

View this table: [in this window] [in a new window]   Table III. TCR CDR3 repertoire in single-cell-sorted Kd-V7+ CD8+ T cells in female NOD mice pre- and post- VRP-IGRP immunization

	Discussion

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The analysis of cell-specific T cell reactivity in at-risk or diabetic individuals is dependent on T cells prepared from blood. However, it is unclear how accurate clonotypes residing in blood reflect those targeting the islets. Insight into this issue would aid in our understanding of disease progression, in addition to the development of novel strategies to monitor the efficacy of immunotherapies applied in the clinic. Accordingly, the current study was conducted to define the TCR repertoire of disease-relevant IGRP_206–214-specific CD8⁺ T cells residing in PBL, islets, and PLN of female NOD mice. CD8⁺ T cells were sorted from the respective tissues using K^d tetramers complexed with the mimetic NRP-V7 peptide, and CDR3 and CDR3 segment heterogeneity was determined at a single-cell level. K^d tetramers complexed with NRP-V7 rather than IGRP_206–214 were used in the study because a broader spectrum of clonotypes was typically detected in K^d-V7⁺ vs IGRP_206–214⁺ CD8⁺ T cells sorted from the same individual NOD mice (C. P. Wong and R. Tisch, unpublished results). Noteworthy is the fact that prevalent clonotypes were shared; however, only a subset of the overall repertoire of K^d-V7⁺ CD8⁺ T cells was detected in K^d-IGRP_206–214⁺ CD8⁺ T cells, likely reflecting the higher binding avidity of K^d-V7 compared with K^d-IGRP_206–214 (C. P. Wong and R. Tisch, unpublished results).

An important observation made in this study is that the same immunodominant clonotypes detected in PBL were also prevalent in the islets (and PLN) of six of eight untreated NOD female mice (e.g., NOD mice #1, #2, and #4–7; Table II), although the frequency of these T cell clones varied depending on the tissue. T cell clonotype distribution was independent of disease status because both diabetic (mice #2, #4, #5) and 20-wk-old nondiabetic (mice #1, #6, #7) NOD female mice contained the same immunodominant clones in PBL and islets (and PLN) (Table II). Untreated NOD mouse #4 provided an extreme example in which K^d-V7⁺ CD8⁺ T cells consisted of a single clonotype (based on both CDR3 and CDR3 motif usage) that was detected in all three tissues (Fig. 2 and Tables I and II). Exceptions to the general trend were nevertheless observed. For instance, in untreated NOD mice #3 and #8, the CDR3 sequences STDWGYEQ and SSLDRVEQ, respectively, were prevalent in PBL but not the islets (Table II). In contrast, the CDR3 sequences SAERGANSDYT and SSGDNYEQ dominated the islets but not PBL in untreated NOD mice #3 and #8, respectively (Table II). Fluctuation in the number of K^d-V7⁺ CD8⁺ T cells residing in PBL may account for differences detected in the few mice in which there was a significant disparity with a T cell clone found in the islets. For example, the number of K^d-V7⁺ CD8⁺ T cells in PBL has been shown to undergo continual rounds of expansion and contraction, which in turn is thought to indicate waves of clonal proliferation of IGRP_206–214-specific CD8⁺ T cells undergoing avidity maturation in the islets and/or PLN (11). Nevertheless, in the majority of untreated NOD mice, K^d-V7⁺ CD8⁺ T cell clonotypes prevalent in PBL represent a "selected" repertoire that is involved in islet-infiltration. This interpretation is consistent with work by Trudeau et al. (11), which demonstrated that detection of K^d-V7⁺ CD8⁺ T cells in PBL of euglycemic NOD mice provides a relatively accurate predictive marker for the progression to overt diabetes. We recently demonstrated that K^d-V7⁺ CD8⁺ T cell clones prevalent in the endogenous islets are also dominant in early infiltrates of islet grafts implanted in diabetic NOD recipients (17). This observation coupled with the findings made in the current study, suggest that T cell clones mediating islet graft destruction are directly recruited from PBL. Further evidence indicating that clonotypes found in PBL exhibit a diabetogenic capacity is provided by Roep and colleagues (24) who showed that cell-specific CD4⁺ T cell clones prepared from PBL of diabetic patients readily traffick to the islets after transfer into NOD.scid mice.

The association between T cell clonotypes residing in PBL and the target tissue is a key issue for several autoimmune diseases including T1D. Addressing this issue has proven to be problematic in large part due to the lack of knowledge of the critical autoantigens and corresponding epitopes driving the respective autoimmune responses. Celiac disease (CD), however, is an exception in which HLA-DQ2-restricted, transglutaminase-modified wheat gliadin peptides have been identified as major targets of CD4⁺ T cells in the intestinal mucosa (25). Evidence indicates that gliadin-specific T cells in PBL vs the gut of CD patients differ in terms of HLA-restriction, epitope specificity, and a requirement for transglutaminase-mediated deamidation of the epitopes (26). Nevertheless, disease-relevant CD4⁺ T cell clones have been reported in PBL of CD patients under certain conditions such as challenging individuals with Ag (27) or using a sensitive CFSE-based assay to measure proliferation in response to deamidated gliadin in vitro (28). These data suggest that in CD and possibly other tissue-specific autoimmune diseases, only a low frequency of pathogenic effectors exists in PBL of patients that may vary depending on disease progression. In contrast, our findings indicate that the frequency of cell-specific CD8⁺ T cells can be relatively high in PBL (i.e., >4% of CD8⁺ T cells) and that these T cell clones are also generally found dominating the islets of NOD mice. An important question that still needs to be addressed, however, is whether CD8⁺ and CD4⁺ T cells specific for other cell autoantigens also exhibit a similar clonotypic distribution between the periphery and islets of NOD mice, and ultimately in T1D patients.

Vaccination of NOD female mice with VRP-IGRP provided further insight into the TCR repertoire of IGRP_206–214-specific CD8⁺ T cells in the respective tissues. First, the repertoire for IGRP_206–214-specific CD8⁺ T cells in the periphery is relatively limited. For example, immunodominant clonotypes detected in PBL before vaccination were also prevalent in the PBL of four of five NOD mice treated with VRP-IGRP despite expansion of up to 40-fold in K^d-V7⁺ CD8⁺ T cells (Fig. 3, A and B). Novel clonotypes were detected in postvaccination PBL of some of the NOD mice (mice #1, #3, #5); however, these T cell clones were found only at a low frequency (Table III). Notably, VRP-IGRP vs VRP-HA vaccination had no significant effect on the number of K^d-V7⁺ CD8⁺ T cells detected in the islets (or PLN), indicating that expansion of IGRP_206–214-specific CD8⁺ T cells occurred predominately in the periphery (e.g., PBL and spleen). Importantly, in five of five mice the same immunodominant clonotypes detected in pre- and postvaccination PBL also were prevalent in the islets and PLN, although again the frequency of these clonotypes varied in the respective tissues (Table III). This observation further supports the conclusion that the repertoire of IGRP_206–214-specific CD8⁺ T cells in PBL is similar to islet-infiltrating T cell clones, even after immunization.

TCR analysis in patients with tissue-specific autoimmune diseases such as rheumatoid arthritis or multiple sclerosis have shown variation in immunodominant clonotypes among individuals, and within the targeted tissues (i.e., joints or brain plaques) of the same individual (29, 30). Interestingly, Carnaud and colleagues (31) have also demonstrated that a high degree of heterogeneity of TCR CDR3 gene usage exists at an early age among islets in NOD mice, although the specificity of these T cells was not determined. These studies are consistent with the high degree of variability detected in CDR3 sequences between individual NOD mice in the current work (Tables II and III) and in an earlier study investigating the repertoire of single K^d-V7⁺ CD8⁺ T cells isolated from grafted vs endogenous islets (17). Interestingly, the CDR3 motif SDSQNTL was detected either as a dominant or minor clone in two of eight untreated (mice #2, #3; Table II), four of five VRP-IGRP-treated mice (mice #2–5; Table III), and in six of eight mice examined in our earlier study (17). The marked heterogeneity observed in the CDR3 segment would suggest that specificity and affinity associated with IGRP_206–214 clonotypes is largely due to the canonical V17-J42 elements (7). However, in view of the relatively high frequency among individual NOD mice, the CDR3 SDSQNTL motif may also contribute to TCR specificity and/or affinity.

This study extends previous work (11, 17) by providing novel insight into the clonotypic composition and distribution of disease-relevant CD8⁺ T cells at the single-cell level in individual, unmanipulated, and immunized NOD mice. Specifically, fine TCR repertoire analysis of single IGRP_206–214-specific CD8⁺ T cells demonstrates that a limited number of immunodominant clonotypes found in PBL typically reside in the islets. Furthermore, clones prevalent in PBL are preferentially expanded following immunization, and it is these clonotypes that typically reside in the islets. Finally, this study shows that insight into the clonotypic nature of disease-relevant T cells (e.g., islet infiltrating) can be gained by analysis of T cells isolated from PBL.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants R01AI058014 and R01AI52435, and Juvenile Diabetes Research Foundation Grant 1-2003-611. C.P.W. was supported by a Juvenile Diabetes Research Foundation postdoctoral fellowship. K.S.G. was supported by a National Institutes of Health training grant.

² Address correspondence and reprint requests to Dr. Roland Tisch, Department of Microbiology and Immunology, Mary Ellen Jones Building, Room 804, Campus Box 7290, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7290. E-mail address: rmtisch{at}med.unc.edu

³ Abbreviations used in this paper: T1D, type 1 diabetes; IGRP, islet specific glucose-6-phosphatase catalytic subunit-related protein; PLN, pancreatic lymph node; VRP, Venezuelan equine encephalitis virus replicon particle; HA, hemagglutinin; CD, celiac disease.

Received for publication May 10, 2006. Accepted for publication November 21, 2006.

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