Assessment of CD4+ T Cell Responses to Glutamic Acid Decarboxylase 65 Using DQ8 Tetramers Reveals a Pathogenic Role of GAD65 121–140 and GAD65 250–266 in T1D Development

<div><p>Susceptibility to type 1 diabetes (T1D) is strongly associated with MHC class II molecules, particularly HLA-DQ8 (DQ8: DQA1*03:01/DQB1*03:02). Monitoring T1D-specific T cell responses to DQ8-restricted epitopes may be key to understanding the immunopathology of the disease. In this study, we examined DQ8-restricted T cell responses to glutamic acid decarboxylase 65 (GAD65) using DQ8 tetramers. We demonstrated that GAD65_121–140 and GAD65_250–266 elicited responses from DQ8+ subjects. Circulating CD4+ T cells specific for these epitopes were detected significantly more often in T1D patients than in healthy individuals after in vitro expansion. T cell clones specific for GAD65_121–140 and GAD65_250–266 carried a Th1-dominant phenotype, with some of the GAD65_121–140-specific T cell clones producing IL-17. GAD65_250–266-specific CD4+ T cells could also be detected by direct ex vivo staining. Analysis of unmanipulated peripheral blood mononuclear cells (PBMCs) revealed that GAD65_250–266-specific T cells could be found in both healthy and diabetic individuals but the frequencies of specific T cells were higher in subjects with type 1 diabetes. Taken together, our results suggest a proinflammatory role for T cells specific for DQ8-restricted GAD65_121–140 and GAD65_250–266 epitopes and implicate their possible contribution to the progression of T1D.</p></div

Direct ex vivo analysis of GAD65-specific T cells.

<p>Unmanipulated PBMCs were stained with DQ8/GAD65_250–266 PE-tetramer. Antigen-specific CD4+ T cells were enriched, stained with antibodies against surface markers of interest, and analyzed on a Calibur multi-color flow cytometer. (<b>A</b>) Representative ex vivo analysis of the surface memory marker CD45RO for GAD65_250–266. The frequency of GAD65_250–266-specific CD45RO+CD4+ T cells was 4.4 per million CD4+ T cells for the T1D patient (left panel) and 0.6 per million for the healthy subject (right panel). (<b>B</b>) Ex vivo co-staining of GAD65_250–266-specific cells with DQ8/GAD65_250–266 PE- and DQ8/GAD65_250–266 APC-tetramers. Cells were stained with PE-labeled DQ8/GAD65_250–266 tetramers first. After enrichment, tetramer-positive cells were stained again with APC-labeled DQ8/GAD65_250–266 tetramers at 37°C for 1 h. (<b>C</b>) Cumulative total CD4+ and CD45RO+CD4+ T cell frequencies for GAD65_250–266 in controls (open circles, n = 10) and T1D patients (closed circles, n = 10). <b>***</b> P<0.001, <b>**</b> P<0.01, as evaluated by Mann-Whitney U-test.</p

List of DQ8-restricted GAD65 peptides used in this study.

<p>*IC₅₀ value for the control H1MP_185–204 peptide was 1.4 µM.</p><p>*Predicted binding registers are indicated in boldface.</p><p>List of DQ8-restricted GAD65 peptides used in this study.</p

Summary of ex vivo results for GAD65_250–266.

<p>N/A: not available.</p><p>Summary of ex vivo results for GAD65_250–266.</p

Prevalence of GAD65-specific T cells in T1D and healthy subjects.

<p>Not all peptides were tested on each subject.</p><p>Statistical analysis was performed using two-tailed Fisher's exact tests.</p><p>Prevalence of GAD65-specific T cells in T1D and healthy subjects.</p

Functional analysis of DQ8-specific self-reactive T cells.

<p>T cell clones isolated by DQ8 tetramers were assayed for specificity and functionality. (<b>A</b>) Tetramer staining for GAD65_121–140-specific (clone T1D05-C2), GAD65_250–266-specific (clone T1D04-C1) and unrelated T cell clones. (<b>B</b>) Representative proliferation results of one GAD65_121–140- and two GAD65_250–266-specific T cell clones using APCs from a DR0401/DQ8 homozygous individual. Cells were stimulated with specific or irrelevant control peptide in the absence or presence of 20 µg/ml of L243 (HLA-DR blocking antibody) or SPVL3 (HLA-DQ blocking antibody). SI: stimulation index; cpm of specific peptide divided by cpm of irrelevant peptide. (<b>C</b>) Summary of cytokine-positive clones (for definition see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112882#s4" target="_blank">Materials and methods</a>) for GAD65_121–140 and GAD65_250–266. T cell clones were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate and 1 µg/mL ionomycin in the presence of 10 µg/mL Brefeldin A in 1 mL of T cell medium for 4 hours at 37°C. Cells were fixed, permeabilized, stained with antibodies for IFN-γ, IL-10, IL-4, and IL-17, and analyzed on a LSRII multicolor flow cytometer. (<b>D</b>) Secretion of IFN-γ, IL-4, and IL-10 from two GAD65_121-140-specific and two GAD65_250–266-specific T cell clones. Clones were stimulated in the presence of 10 µg/ml of specific peptide with DQ8 antigen presenting cells.</p

Differences in T cell number and phenotype for subjects with asymptomatic or neuroinvasive WNV infection.

<p>A) WNV-specific T cells from a total of 11 asymptomatic subjects (open symbols) and 9 subjects with neuroinvasive infection (filled symbols) as enumerated by ex vivo tetramer staining. Each data point represents tetramer staining for a single WNV epitope (2–3 epitopes were measured per subject) measured in a single subject. Subjects with neuroinvasive infection has significantly more WNV specific T cells (p<0.0001). B) Comparison of the relative proportion of naïve (CD45RA+CCR7+), TEM (CD45RA-CCR7-), TCM (CD45RA-CCR7+), and TEMRA (CD45RA+CCR7-) WNV specific T cells as determined by ex vivo tetramer analysis. Subjects with neuroinvasive infection (filled symbols) had a significantly lower proportion of naïve WNV specific T cells than asymptomatic subjects (open symbols) (p < 0.05) and a significantly higher proportion of TEM. C) Comparison of the relative proportion of WNV specific T cells within various defined functional subsets. Subjects with neuroinvasive infection had a significantly lower proportion of WNV specific T cells that were Th1-like (CXCR3+CCR4-CCR6-) (p < 0.0001) than asymptomatic subjects (p < 0.05) and a significantly higher proportion of Th1^ (CXCR3+CCR4+CCR6-) cells (p < 0.0001). D) Comparison of intracellular IFN-γ, IL-4, and IL-17 staining for WNV specific T cell lines from asymptomatic subjects (open symbols) or subjects with neuroinvasive infection (filled symbols). A significantly higher percentage of WNV specific T cells from subjects with neuroinvasive infections were positive for IL-4 (p<0.05). All other cytokines were not significantly different.</p

Comparing the absolute number of various subsets of WNV specific T cells in WNV subjects with neuroinvasive versus asymptomatic infection.

<p>A) Neuroinvasive subjects (9 subjects, designated by filled symbols) had higher numbers of WNV specific CD4+ T cells that were TEM or TCM (p < 0.0001 and p < 0.01 respectively) than asymptomatic subjects (11 subjects, designated by open symbols). B) Neuroinvasive subjects (filled symbols) had higher numbers of WNV specific CD4+ T cells that were Th1 or Th1^ (p < 0.0001 and p < 0.01 respectively) than asymptomatic subjects (open symbols).</p

Subjects with neuroinvasive WNV infections have an atypically polarized cytokine response.

<p>The large pie charts depict the proportion of WNV specific T cell lines obtained from 8 subjects with neuroinvasive infections (upper panel) or 8 subjects with asymptomatic infections (lower panel) that stained positive for IFN-γ only, IL-4 only, IL-10 only and IL-17 only, or any combination of these cytokines ('multiple'). Within the 10.7% and 4.1% of T cells respectively that produced multiple cytokines, the small pie charts indicate the proportion of cells in subjects with neuroinvasive infections (upper panel) or asymptomatic infections (lower panel) that stained positive for IFN-γ and IL-4; IFN-γ and IL-17; IL-4 and IL-17; or IFN-γ, IL-4, and IL-17. For all other possible combinations the percentage observed was negligible. A significantly higher percentage (p<0.0001) of WNV-specific T cell lines isolated from subjects with neuroinvasive infections were positive for more than one cytokine than from subjects with asymptomatic infections The elevated percentage of WNV-specific T cells that produced dual cytokines in subjects with neuroinvasive infections was almost exclusively due to T cells that co-produced IFN-γ and IL-4. These cells occurred at a significantly higher proportion (p<0.0001) in subjects with neuroinvasive infections than in subjects with asymptomatic infections whereas the proportion of T cells that co-produced other combinations of cytokines did not differ.</p

Altered Treg phenotype in subjects with neuroinvasive WNV.

<p>A) CD4+ Tregs were evaluated in PBMC samples from 9 subjects with neuroinvasive infection or 8 subjects with asymptomatic WNV by gating on CD25+CD4+ T cells and subsequently gating on CD127lowFoxP3++ T cells (designated by the upper left rectangle). B) Each symbol represents the overall percentage of Treg (CD4+CD25+CD127lowFoxP3++ T cells) within the PBMC of a single WNV infected subject. The percentage of CD4+ Tregs was not significantly different between subjects with neuroinvasive or asymptomatic WNV. C) Each symbol represents the percentage of Treg that were CTLA-4 positive for a single WNV infected subject. Subjects with neuroinvasive WNV had a significantly lower percentage of CTLA-4 positive Tregs (p = 0.0097). D) Each symbol represents the percentage of Treg that were CCR4 positive for a single WNV infected subject. The percentage of Treg that were CCR4+ was not significantly different between subjects with neuroinvasive or asymptomatic WNV.</p