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* Telethon-Juvenile Diabetes Research Foundation Center for Beta Cell Replacement, San Raffaele Scientific Institute, Milan, Italy;
Department of Pediatrics, San Raffaele Scientific Institute, Milan, Italy; and
Diabetes Research Institute, Munich, Germany
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Abstract |
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cell autoantigen-reactive T cells that are involved in the pathogenesis of type 1 diabetes has been troublesome for many laboratories. Disease-relevant autoreactive T cells should be in vivo Ag experienced. The aim of this study was to test this hypothesis and then use this principle as a strategy for identifying diabetes-relevant autoreactive T cells. In this study, a CSFE dilution assay was used to detect glutamic acid decarboxylase 65 (GAD65)- and insulin-responsive T cells and HLA-0201*-GAD65114–122 pentamers were used to detect CD8+ GAD-responsive T cells in memory CD45RO+ and naive CD45RO– cell populations from patients with type 1 diabetes and healthy control subjects. T cell proliferative history was evaluated by flow cytometry telomere length measurement. CD4+ and CD8+ T cells specific for GAD65 and insulin were present in patients with type 1 diabetes and control subjects. Within the naive CD45RO– cells, CD4+ and CD8+ T cell responses were similar between patients and controls. Within the memory CD45RO+ cells, CD4+ T cell responses against whole GAD65 and insulin and HLA-0201*-GAD65114–122 pentamer-positive CD8+ T cells were found in patients with type 1 diabetes, but not in control subjects (p < 0.05 for all). Responding cells from the CD45RO+ T cell population had substantially shorter telomere lengths than responding cells from the CD45RO– cell population. Diabetes-specific autoreactive T cells in the circulation have uniquely undergone sustained in vivo proliferation and differentiation into memory T cells. Prior selection of these cells is possible and is a way to identify diabetes-relevant target Ags and epitopes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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cells mediated by autoreactive T cells (1, 2, 3). Several cell autoantigens are targets of islet autoimmunity. Of these, glutamic acid decarboxylase 65 (GAD65), insulinoma-associated protein 2, and (pro)insulin appear to be highly antigenic in humans both for T cells (4, 5, 6) and B cells (7, 8). To date, immune markers for T1DM have primarily centred on the presence of autoantibodies to cell Ags and measurement of these Abs has been shown to be useful for prediction and diagnosis of type 1 diabetes (9). In contrast, T cell responses to islet cell Ags have been inconsistent in their diabetes specificity. Early studies reported that GAD65 and (pro)insulin-specific T cell responses were preferentially detectable in patients with T1DM and at-risk subjects, but rarely in healthy individuals (10, 11, 12). Other studies, however, showed that T cell responses against T1DM-associated autoantigens can be readily measured both in patients with T1DM and subjects without any sign of autoimmunity (13).
Recent studies indicate that autoreactive T cells in patients with type 1 diabetes can be distinguished by characteristics that are typical of cells that have already encountered Ag. These include proliferation to Ag in the absence of costimulatory signals (14) and the presence of specific late activation markers (15). These studies suggest that it may be possible to uniquely identify autoreactive T cell responses in at-risk subjects and patients using assays that selectively measure memory T cell responses. To test this hypothesis, we have investigated CD4 and CD8 T cell responses to autoantigen using whole PBMC, CD45RA+, and CD45RO+ T cell subsets. To test the hypothesis that autoreactive T cells in patients have undergone expansion in vivo, we have estimated proliferative history using telomere length. In human germline cells, telomeres are 
20-kb long (16) and most cells, including leukocytes, undergo the loss of 50–100 bp at each cell division (17) as a result of the balance between telomere erosion during cell division and the activity of telomerase, a unique reverse transcriptase that has the ability to extend the 3' end of telomeres (18, 19). Telomeres consist of hexanucleotide repeats, and telomere length can be determined by hybridization of fluorescence peptide nucleic acid probes to these repeat sequences (19). By separately studying autoantigen-induced proliferation in CD45RA+ and CD45RO+ T cell subsets and by examining the telomere length of proliferating cells, we show that the autoantigen-reactive T cells from at-risk subjects and patients have an Ag-experienced in vivo proliferation history and in this regard are distinct from those in healthy subjects.
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Materials and Methods |
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Patients with recent (<6 mo) onset T1DM, Ab-positive at-risk subjects, and healthy donors were recruited at the Department of Pediatrics (San Raffaele Scientific Institute, Milan, Italy) and at the Diabetes Research Institute (Munich, Germany). A total of 68 subjects including 35 patients with T1DM (median age, 14 years; range, 8–29 years; 18 males) 13 Ab-positive at-risk subjects (median age, 18 years; range 2–62 years; 5 males), and 20 healthy control subjects (median age, 15.6 years; range, 7–40 years; 9 males) were provided as blind-coded samples for testing. Peripheral venous blood samples were obtained by venipuncture with informed consent and ethics committee approval. Patients characteristics are shown Table I.
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PBMC were isolated by Ficoll-Hypaque (Amersham Pharmacia Biotech) density gradient centrifugation from sodium-heparinized venous blood samples and washed twice in PBS.
CSFE proliferation assay
Cells were cultured in RPMI 1640 (BioWhittaker) supplemented with 10% pooled human serum (Sigma-Aldrich), 2 mM glutamine (Glutamax; Invitrogen Life Technologies), penicillin (100 U/ml), and streptomycin (100 U/ml; Invitrogen Life Technologies), and 100 µM nonessential amino acids (Invitrogen Life Technologies). Proliferation was determined using the CFSE (Molecular Probes) dilution assay as previously described (20). PBMC at 107/ml in PBS were incubated at 37°C for 5 min with 0.2 µM CFSE. Staining was terminated by adding RPMI 1640 containing 5% pooled human AB serum (Sigma-Aldrich) at 4°C, followed by one wash in PBS. Stained cells were cultured at 2 x 105/well in 100 µl of culture medium in 96 round-bottom plates in a standard 37°C CO2 incubator for 1 wk. PBMC cultures were stimulated with 5 µg/ml recombinant human GAD65 (Diamyd Diagnostic), recombinant human insulin (10 µg/ml; Serologicals) or tetanus toxoid (TT; 40 level of flocculation units/ml; Connaught Lab).
CFSE dilution assay on separated CD4+/CD45RO+ and CD4+/CD45RO– cells
Separation of CD4+/CD45RO+ and CD4+/CD45RO– cells was performed in two steps using magnetic microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD4+ cells were separated from PBMC using a CD4 multisort kit and the CD4+ fraction was further separated into CD4+CD45RO+ and CD4+CD45RO– using a CD45RO sort kit. The CD4– fraction containing monocytes and B cells for optimal Ag presentation was also collected. CD4+CD45RO+ and CD4+CD45RO– cells were mixed 1:1 with the CD4– fraction. Mixed cells were all labeled with CFSE and cultured with Ags as previously described (20).
[3H]Thymidine incorporation assay
In some experiments, we used a proliferation assay based on [3H]thymidine incorporation. CD4+CFSEdim cells were mixed 1:1 with autologous irradiated (3000 rad) PBMC and cultured for 5 days with Ags at the concentrations used for CFSE dilution assay. [3H]Thymidine(1 µCi/ml) was added for the last 14 h.
Pentamer staining
Pentamer staining was performed both on fresh PBMC and on PBMC stimulated in vitro for 10 days in culture medium with the immunogenic GAD65114–122 peptide. The GAD65114–122 peptide has been demonstrated to elicit CTLs in patients with T1DM (21). The following pentamers all purchased from ProImmune were used: HLA*0201-GAD65114–122 (VMNILLQYV), HLA-A*0201-influenza A matrix protein58–66 (GILGFVFTL), and HLA-A*0201-HIV-1 gag p17 76–84 (SLYNTVATL). For class I pentamer staining, freshly isolated or cultured PBMC were washed twice and resuspended at 107 cells/ml in X-vivo 15 medium (BioWhittaker) supplemented with 10% pooled human serum (Sigma-Aldrich). Three hundred microliters of cell suspension were then incubated for 30 min at room temperature in the dark with 0.5 µg/ml pentamers, 1 µg/ml anti CD8 PE-Cy5 (mouse IgG1k, clone RPA-T8; BD Pharmingen), and 1 µg/ml anti CD45RO-allophycocyanin (clone IgG2a, 
, clone UCHL-1; BD Pharmingen). For FACS analysis, cells were gated as live cells based of the forward scatter (FSC) and side scatter (SSC) parameters. Gating of lymphocytes on FSC and SSC was used to minimize nonspecific staining. A total of 5 x 105 live lymphocytes were acquired from each sample.
FACS analysis
For the CFSE dilution assay, cells were harvested after 7 days of culture, washed in PBS, and stained on ice with an anti-human CD4 PerCP (mouse IgG1, clone SK3; BD Pharmingen). Optimal compensation and gain settings were determined for each sample based on unstained and single-stained samples. Propidium iodide was used to exclude dead cells. Ten thousand propidium-iodide negative, lineage-positive cells were acquired from each sample. The number of cells that had proliferated was determined by gating on the lineage-positive, CFSEdim subset. Data were collected with a FACSCalibur flow cytometer and analyzed with the WinMDI software (Stanford University, Stanford, CA).
Telomere length measurement by flow cytometry
Telomeres consist of noncoding G-rich hexanucleotide repeats (TTAGGG)n and associated proteins. We used a carboxylfluorescein-conjugated peptide nucleic acid probe (PNA; Primm) specific for telomere sequences that hybridized in a quantitative way to telomere repeats. The sequence of the carboxylfluorescein-labeled PNA probe was AATCCCAATCCCAATCCC. For staining, 3 x 105 cells were resuspended 300 µl of hybridization solution (Hybmix: 75% formamide, 20 mM Tris buffer, 1% BSA, 20 mM sodium chloride, water) containing 0.3 µg/ml PNA probe and incubated for 10 min at room temperature. Samples were subjected to heat denaturation at 80°C for 15 min followed by hybridization at room temperature for 90 min. Samples were washed two times in Hybmix and PBS, respectively. As heat denaturation at 80°C also denaturates proteins, it is not possible to stain cells with Abs. Thus, the use of a FITC probe does not permit the use of CFSE as they have overlapping emission spectra. To overcome these problems, PBMC were separated into CD4+CD45RO+ and CD4+CD45RO– cells, labeled with the lipophilic tracer DiD Vibrant cell labeling solution (Molecular Probes), and stimulated with Ags. After 7 days, the CD4+CD45RO+DiDdim and CD4+CD45RO–DiDdim cells were FACS sorted and telomere lengths were measured. For DiD Vibrant cell labeling, cells were suspended at a density of 106/ml in serum-free RPMI 1640 plus 5 µl/ml DiD Vibrant solution for 15 min at room temperature in the dark. Cells were subsequently washed two times in RPMI 1640 supplemented with 10% FSC. As for CFSE, DiD Vibrant fluorescence is reduced by half in daughter cells upon each cell division and can be alternatively used with fluorochromes with emission spectra overlapping those of CFSE.
Statistical analysis
To evaluate the CD4+CFSEdim proliferative response in patients with T1DM and healthy subjects, the median percentage of CD4+CFSEdim cells and interquartile ranges (IQR) were calculated for each group. The Mann-Whitney U test was used to compare responses of patients with T1DM and healthy controls to the same Ag. For all analyses, a two-tailed p value of 0.05 was considered significant. Statistical analyses were performed using the Statistical Package for Social Science (SPSS 11.0).
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Results |
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Proliferative responses to GAD65 and insulin were measured by the CFSE dilution assay. First, the capacity of the CFSE dilution assay to detect Ag specific T cell proliferation was assessed (Fig. 1A). PBMC were labeled with CFSE and stimulated for 1 wk with GAD65 (5 µg/ml), insulin (10 µg/ml), and TT (40 LFU/ml). CD4+CFSEdim cells were detected for GAD65 (3.2%), insulin (2.7%), and the recall Ag TT (9.1%) as compared with negative control background (0.2%). To evaluate whether CD4+CFSEdim cells were enriched for Ag-specific T cells, CD4+CFSEdim cells obtained from stimulation with GAD65 and insulin were FACS sorted and then restimulated with the same Ag used in the first stimulation or with another Ag. [3H]Thymidine incorporation was used to measure proliferation (Fig. 1B). CD4+CFSEdim obtained from GAD65-stimulated cells responded to rechallenge with GAD65 but not insulin. Similarly, CD4+CFSEdim obtained from insulin-stimulated cells responded to rechallenge with insulin but not GAD65. These data suggest that the CFSE dilution assay detects GAD65- and insulin-specific CD4+ T cell responses in vitro.
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Proliferative responses to GAD65 and insulin were measured in blinded samples from 43 subjects (Fig. 1C). No differences were observed between patients and control subjects with respect to the percentage of CD4+CFSEdim cells in the absence of Ag (T1DM: median, 0.3%; IQR, 0.15–0,5%; vs controls: median 0.3%; IQR, 0.3–0.5%; p = 0.9), the percentage of CD4+CFSEdim cells after incubation with GAD65 (T1DM: median, 1,7%; IQR, 1.05–3,05%; vs controls: median 1.7%; IQR, 0.3–2.9%; p = 0.6), and the percentage of CD4+CFSEdim cells after incubation with insulin (T1DM: median, 1,05%; IQR, 0.65–1,75%; vs controls: median 0.5%; IQR, 0.3–2.05%; p = 0.3). These results provide evidence that autoreactive T cells responding to GAD65 and insulin are present both in patients with T1DM and in healthy control subjects.
Autoreactive CD4+ T cells have a memory phenotype in patients and at-risk subjects but not in control subjects
We reasoned that if GAD65 and insulin-reactive T cells were involved in the disease process, they should have been activated during the course of the disease and could have a memory T cell phenotype. Thirteen patients with T1DM (subject code numbers 003, 005, 006, 015, 018, 020, 028, 044, 046, 047, 050, 051, 053), 13 Ab-positive at-risk subjects (subject code numbers 056 to 068), and 13 age- and sex-matched healthy controls (subject code numbers 007, 009, 022, 026, 031, 040, 041, 045, 048, 049, 052, 054, 055) were selected for separation into naive and memory T cells. Naive CD4+ T cells are CD45RA+/CD45RO– while memory T cells are CD45RA–/CD45RO+. Because all CFSEdim cells after 1 wk of stimulation with Ag express CD45RO (data not shown) CD4+/CD45RO+ and CD4+/CD45RO–, cells were isolated immediately after PBMC isolation and the two subsets were stimulated separately (Fig. 2A).
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To corroborate the findings of an Ag-experienced autoreactive memory T cell population in the peripheral blood of patients with T1DM and at-risk subjects, we examined telomere length of the proliferating memory and naive cells in 8 patients with T1DM (005, 006, 015, 018, 020, 028, 032, and 034), 13 at-risk subjects (from 056 to 068), and eight healthy controls (007, 009, 022, 026, 031, 039, 040, 041) (Fig. 4). If autoreactive T cells from diabetic individuals had been primed in vivo to differentiate into CD45RO+ memory T cells, they were likely to have undergone cell division and therefore have reduced telomere length. Carboxylfluorescein-labeled PNA probes were used to measure telomere length by flow cytometry at the single-cell level (19). Because carboxylfluorescein and CFSE have similar emission spectra, PBMC were stained with the carbocyanine dye DiD Vibrant cell labeling solution. CD4+CD45RO+ and CD4+CD45RO– were FACS sorted (Fig. 4A) and a 7-day proliferation assay in the presence of GAD65, insulin, or TT was performed as above. Telomere length was measured in sorted CD4+/DiDdim cells in a subset of subjects (Fig. 4B).
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GAD65-specific CD8+ T cells with a memory phenotype in patients with T1DM
Because we were able to detect T1DM-specific CD4+ T cell response phenotypes, we examined CD8+ T cell responses to a previously reported GAD65 epitope (21). HLA-A*0201-restricted GAD65114–122 pentamers were used to detect GAD65-specific CD8+ T cells. HLA-A*0201 influenza A matrix protein58–66 pentamers were used as a positive control and HLA-A*0201 HIV-1 gag p1776–84 pentamers were used as a negative control (all individuals tested were seronegative for HIV-1).
GAD65114–122-specific CD8+ T cells were abundant in PBMC stimulated in the presence of GAD65114–122 peptide for 10 days in both patients and control subjects (Fig. 5A). We therefore examined tetramer binding in fresh unstimulated, uncultured PBMC (Fig. 5, B and C). Influenza A matrix protein58–66-specific CD8+ T cells were observed in all individuals with similar frequencies in patients, at-risk subjects, and controls (median 0.32% of CD8+ T cells in T1DM vs median 0.31% in Ab+ vs median 0.33% in controls). T cells specific for the HIV-related peptide gag p1776–84 were not detected. GAD65114–122-specific CD8+ T cells were detected in all subjects studied (median 0.24% of CD8+ T cells in T1DM vs 0.24% in at-risk subjects vs 0.35% in controls). Consistent with CD4+ T cell data, GAD65114–122-specific CD8+ T cells were observed in the CD45RO– subset from patients, at-risk subjects, and control subjects whereas GAD65114–122-specific CD8+ T cells in the CD45RO+ subset were significantly increased in patients as compared with control subjects (p = 0.03 T1DM vs controls; p = 0.012 Ab+ vs control, Fig. 5C) and were also observed in the at-risk subjects.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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cell autoantigens are here demonstrated in the peripheral blood of both healthy individuals at-risk subjects and patients with T1DM. Unique to patients with T1DM and at-risk subjects were autoantigen responsive T cells that had been activated and expressed CD45RO and had undergone substantial cell division in vivo. The findings support the notion that autoantigen-responsive T cells are part of the normal T cell repertoire, that cell autoantigen-responsive T cells are relevant to the disease process in T1DM, and that before and by the time of clinical disease onset these cells have received stimuli consistent with Ag-induced proliferation.
T cell responses to GAD65 and to insulin have been repeatedly shown in patients with T1DM. Responses have been inconsistently demonstrated to be more frequent in patients than in control subjects (22). Recent reports have shown that the T cells responding to cell autoantigens and their peptides are phenotypically different to those in control subjects. Patients have proinsulin peptide-responsive T cells that produce IFN-
whereas those in control subjects usually produce IL-10 (23). GAD65-reactive T cells in patients do not require costimulation indicating that they are Ag-experienced (14) and autoreactive memory T cells from patients have unique coexpression of CD25 and the late activation marker CD134 (15). In a recent report, Danke et al. (24) using class II tetramers showed that GAD65555–567-specific T cells are present only in the CD45RA+ fraction in healthy subjects and both in CD45RA+ and CD45RO+ fraction in patients with T1DM. Our findings are consistent with each of these reports because they all highlight that the normal T cell repertoire includes potentially autoreactive cells and that patients have cells that have encountered and responded to Ag. Like these and other studies, we were unable to see differences in GAD and insulin-responsive cells between patients and control subjects unless we considered T cell phenotypes consistent with prior Ag priming.
One caveat in all studies that examine peripheral blood T cell responses in vitro is that culture assays have high Ag concentration and spatial restrictions. Such conditions may artificially break tolerance in vitro and the in vitro findings may not be operative in vivo. Moreover, such conditions may favor response to low-affinity noncognate Ag. We sought, therefore, to determine the proliferative history of the responding cells. As priming of naive T cells is accompanied by proliferation we measured telomere length of autoreactive T cells. Consistent with a prior history of proliferation, we found that memory T cells that proliferated in response to GAD65 and insulin had shorter telomere length as compared with the naive counterpart in the same subjects. Telomere shortening of 100 arbitrary units of fluorescence has been shown to correspond to a loss of 1500 kb (19). As the telomere loss in T cells has been calculated to be 50–100 bp at each cell division, we can estimate that autoreactive memory cells have undergone 15–30 divisions more than the naive cells before sorting. In vitro proliferation is not expected to be more than four cell divisions and is likely to be similar in the memory and naive T cells populations. Thus, the majority of the divisions that have led to telomere shortening must have occurred in vivo. This provides evidence that the proliferative responses observed in the CD4+CD45RO+ memory cells are representative of the in vivo immune response.
A possible consequence of telomere shortening in T cells undergoing repeated antigenic stimulation and replication is replicative senescence due to excessive telomere erosion. In terms of arbitrary units of fluorescence, autoreactive T cells appear to have longer telomeres than memory T cells responding to TT. This implies that they probably still retain a considerable proliferative potential and the autoimmune response is not exhausted at the time of disease onset. Patients with T1DM also had autoreactive T cells with a naive phenotype and longer telomere length. Although these could potentially be recruited and involved in islet cell destruction, they can also be differentiated into different T cell subsets (25), thereby providing therapeutic value. It is possible to differentiate naive autoreactive T cells into CD4+CD25+ T regulatory cells (26), for example, and the presence of naive GAD and insulin-responsive cells in patients offers the potential to develop autoantigen-specific T reg cell therapy (27). In this context, it is important to determine whether the naive Ag-responding cells are truly responding to cognate Ag or are simply responding as a result of low-affinity T cell cross-reactivity in the presence of high Ag concentration.
On the basis of findings with the CD4+ T cells, we examined whether CD8+ T cells could also be detected and had similar phenotypes. As a model, we used class HLA-A*0201-GAD65114–122 pentamers to detect CD8+ T cells specific for GAD65. The peptide GAD65114–122 has been described as immunogenic peptide restricted for HLA-A*0201 able to trigger a cytotoxic response in patients with T1DM (21). Here, we show as a proof of principle in a limited number of subjects that it is possible to detect diabetes-specific GAD65114–122-specific CD8+ T cells in peripheral blood. We were able to find pentamer-positive cells directly from fresh unstimulated PBMC suggesting a relatively high frequency of T cells with this specificity. Similar to GAD65-specific CD4+ T cells, GAD65114–122-specific CD8+ T cells exist with a naive phenotype in healthy individuals and with a naive and memory phenotype in patients with T1DM.
In conclusion, our data suggest that although T cells specific for GAD65 and insulin are present in the circulation of both patients and healthy subjects, a key step for the pathogenesis of T1DM is the proliferation of autoreactive T cells in vivo and their differentiation into memory clones. The findings provide potential avenues for identifying, measuring, and monitoring autoreactive T cells responses in T1DM and suggest that the naive autoreactive T cell component of patients is an opportunity for immunomodulating therapy.
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Disclosures |
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Footnotes |
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1 This work was supported by Telethon Italy and the Juvenile Diabetes Research Foundation (JT-01, 1-2006-665), and by the Italian Ministry of Health (RF2004 N. 123). P.M. is a fellow of the Vita e Salute San Raffaele University PhD Programme in Molecular Medicine. 
2 Address correspondence and reprint requests to Dr. Ezio Bonifacio at the current address: Center for Regenerative Therapies-Dresden, Dresden University of Technology, Tatzberg 47/49, 01307 Dresden, Germany. E-mail address: ezio.bonifacio{at}crt-dresden.de
3 Abbreviations used in this paper: T1DM, type I diabetes mellitus; GAD65, glutamic acid decarboxylase 65; PNA, peptide nucleic acid probe; IQR, interquartile range; FSC, forward scatter; SSC, side scatter; MFI, mean fluorescence intensity; TT, tetanus toxoid; LFU, level of flocculation units.
Received for publication February 23, 2007. Accepted for publication August 23, 2007.
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) in human prediabetes. J. Immunol. 172: 3955-3962. This article has been cited by other articles:
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  A. L. Putnam, T. M. Brusko, M. R. Lee, W. Liu, G. L. Szot, T. Ghosh, M. A. Atkinson, and J. A. Bluestone Expansion of Human Regulatory T-Cells From Patients With Type 1 Diabetes Diabetes, March 1, 2009; 58(3): 652 - 662. [Abstract] [Full Text] [PDF]
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P. Monti, M. Scirpoli, P. Maffi, L. Piemonti, A. Secchi, E. Bonifacio, M.-G. Roncarolo, and M. Battaglia Rapamycin Monotherapy in Patients With Type 1 Diabetes Modifies CD4+CD25+FOXP3+ Regulatory T-Cells Diabetes, September 1, 2008; 57(9): 2341 - 2347. [Abstract] [Full Text] [PDF]
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E. Martinuzzi, G. Novelli, M. Scotto, P. Blancou, J.-M. Bach, L. Chaillous, G. Bruno, L. Chatenoud, P. van Endert, and R. Mallone The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment Diabetes, May 1, 2008; 57(5): 1312 - 1320. [Abstract] [Full Text] [PDF]
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). Comparisons (p value) were made using the Mann-Whitney U test.
