Figure 1.
Cytokine secretion profiling of islet-antigen-specific T cells by the Direct assay in healthy adults.
PBMCs obtained from ten healthy adults (HS#1–10) were cultured for 48 h with GAD65, ZnT8, and PPI 15-mer peptide clusters (GAD65 C1-C14, ZnT8 C1-C16, and PPI C1-C3). Amounts of IL-2, IL-10, IL-13, IL-17A, and IP-10 in the supernatants were measured by a multiplex bead-based cytokine assay. Cytokine data were transformed into heat-map format indicating the fold increase from the background. Blue indicates the strongest cytokine secretion, whereas yellow indicates the background.
Table 1.
Numbers of peptides and peptide clusters.
Figure 2.
Identification of the epitopes in the Direct assay.
CFSE-labeled PBMCs were cultured with single peptides from a “positive” peptide cluster for 8 days. Secreted cytokines were measured on day 2. CD4+/CD8+ T cell proliferation was assessed on day 8 based on the CFSE dilution. (A) Identification of GAD65-specific T cell epitope in HS#4. GAD65 p#73 from peptide cluster #8 (C#8) was found to induce IP-10 secretion. (B) Assessment of CD4+ T cell proliferation in response to GAD65 p#73 in HS#4. (C) Identification of ZnT8-specific T cell epitopes in HS#2. ZnT8 p#2, p#18, p#33, and p#93 were found to induce IP-10 secretion. (D) Assessment of CD4+ T cell proliferation in response to ZnT8 p#2, p#18, p#33, and p#93 in HS#2. (E) Cytokine secretion and T cell proliferation data in the experiments shown in (D) are shown in a heat-map format.
Table 2.
Summary of the islet-antigen-specific T cell responses in the Direct assay.
Figure 3.
Detection of ZnT8-specific Th1 cells by intracytoplasmic cytokine expression assay.
PBMCs were stimulated with the identified peptides in the presence of anti-CD28/CD49d antibody and brefeldin A. After 6 h stimulation, the expression of intracytoplasmic cytokines was analyzed by flow cytometry. Gated to CD3+CD4+ cells. (A) Expression of IFN-γ and IL-2 in CD4+ T cells (right) and IFN-γ in CD4+CD45RO+ memory T cells (left) specific for ZnT8 peptides in HS#4. (B) Expression of IFN-γ and IL-2 in CD4+ T cells (right) and IFN-γ in CD4+CD45RO+ memory T cells (left) specific for ZnT8 epitopes in HS#7.
Figure 4.
Cytokine secretion profiling of specific CD4+ T cells by the Cytokine-driven assay.
(A) Cytokine secretion by ZnT8 p65-specific T cells in HS#4. Results from the Direct assay. (B) ZnT8 p65-specific CD4+ T cell proliferation in HS#4. (C–D) The Cytokine-driven assay. CFSE-labeled PBMCs from HS#4 were cultured with single peptides from ZnT8 C#11 for 7 days in the presence of IL-2 (day 2–7). (C) Cytokine secretion during 24 h re-stimulation with peptides. (D) ICS assay after 6 h re-stimulation in the presence of brefeldin A. Gated to CFSE-diluted CD4+ T cell populations. (E-F) Cytokine secretion profiling of islet-antigen-specific CD4+ T cells in healthy adults by the Cytokine-driven assay. (E) Cytokine secretion during 24 h re-stimulation with peptides. (F) ICS assay after 6 h re-stimulation in the presence of brefeldin A.
Table 3.
Summary of cytokine secretion profiles in the Cytokine-driven assay.
Figure 5.
Islet-antigen-specific T cells identified by the Direct assay display higher avidities.
(A) Left: The results of the Direct assay and the Cytokine-driven assay with ZnT8 single peptides from C#11 in HS#7. Right: PBMCs were cultured with titrated concentrations of the indicated peptides (0.04–25 µM) in the presence of IL-2 (days 2–7), and re-stimulated cells at a constant concentration (25 µM). IFN-γ secretion after re-stimulation. (B) Left: The results of the Direct assay and the Cytokine-driven assay with GAD65 single peptides from C#10 in HS#2 (top) and with ZnT8 single peptides from C#11. Right: PBMCs were cultured with titrated concentrations of the indicated peptides in the presence of IL-2 (days 2–7), and re-stimulated cells with 25 µM peptide. IL-13 secretion after re-stimulation.
Table 4.
Clinical characteristics of T1D patients and HLA-matched controls.
Figure 6.
Cytokine secretions from ZnT8-specific T cells in T1D patients and healthy adults in the Direct assay.
PBMCs from 15 T1D patients and 15 age/gender/HLA-matched controls were stimulated with 2.5 µM of ZnT8 single peptides (p#2, p#18, p#65, and p#68) in duplicates for 48 hours and six cytokines secreted during the stimulation were measured. (A) Representative results of ZnT8-specific IL-10 and IP-10 secretion with T1D patient samples. (B) Summary of cytokine secretion patterns. The cultures that scored positive in any cytokines were selected. Data was transformed into a heat-map format indicating the fold increase from the background.
Table 5.
Number of positive cytokine responses induced by 2.5 µM of ZnT8 peptides.
Figure 7.
Distinct cytokine expression patterns of ZnT8 specific CD4+ T cells between T1D patients and healthy adults.
PBMCs from 15 T1D patients and 15 age/gender/HLA-matched controls were cultured with ZnT8 single peptides for 7 days in the presence of IL-2 (day 2–7). ICS assay was performed after 6 h re-stimulation with the same peptides. Gated to CD4+ T cells. (A) Representative results of cytokine expressions by CD4+ T cells specific for ZnT8 single peptides in T1D patients and controls. (B, C) Cytokine expression patterns of ZnT8-specific CD4+ T cells. 24 from 60 control PBMC cultures (15 subjects×4 peptides) and 22 from 60 T1D PBMC cultures, which contained CD4+ T cells expressing any of three cytokines at a frequency of >0.5%, were used for the analysis. Statistical significance between control group and T1D group was tested by Student’s t-test. (D) Composition of ZnT8-specific CD4+ T cells with distinct cytokine-expression pattern in T1D patients and controls.