Mapping T Cell Responses to Native and Neo-Islet Antigen Epitopes in at Risk and Type 1 Diabetes Subjects

Abstract

Aims: Recent studies highlight the potentially important role of neoepitopes in breaking immune tolerance in type 1 diabetes. T cell reactivity to these neoepitopes has been reported, but how this response compares quantitatively and phenotypically with previous reports on native epitopes is not known. Thus, an understanding of the relationship between native and neoepitopes and their role as tolerance breakers or disease drivers in type 1 diabetes is required. We set out to compare T cell reactivity and phenotype against a panel of neo- and native islet autoantigenic epitopes to examine how this relates to stages of type 1 diabetes development.

Methods: Fifty-four subjects comprising patients with T1D, and autoantibody-positive unaffected family members were tested against a panel of neo- and native epitopes by ELISPOT (IFN-γ, IL-10, and IL-17). A further subset of two patients was analyzed by Single Cell Immune Profiling (RNAseq and TCR α/β) after stimulation with pools of native and neoepitope peptides.

Results: T cell responses to native and neoepitopes were present in patients with type 1 diabetes and at-risk subjects, and overall, there were no significant differences in the frequency, magnitude, or phenotype between the two sets of peptide stimuli. Single cell RNAseq on responder T cells revealed a similar profile in T1D patients stimulated with either neo- or native epitopes. A pro-inflammatory gene expression profile (TNF-α, IFN-γ) was dominant in both native and neoepitope stimulated T cells. TCRs with identical clonotypes were found in T cell responding to both native and neoepitopes.

Conclusion/interpretation: These data suggest that in peripheral blood, T cell responses to both native and neoepitopes are similar in terms of frequency and phenotype in patients with type 1 diabetes and high-risk unaffected family members. Furthermore, using a combination of transcriptomic and clonotypic analyses, albeit using a limited panel of peptides, we show that neoepitopes are comparable to native epitopes currently in use for immune-monitoring studies.

Keywords: T cell receptor; T cells; autoantibody; cytokines; genes; neoepitopes; transcriptome; type 1 diabetes.

Figure 1

Cytokine T cell responses were measured by ELISPOT, and data are expressed as SI [stimulation index (mean spot number of test peptide/mean spot number of diluent)]. (A) Magnitude of IFN-γ (red) IL-10 (blue) and IL-17 (purple) responses in preclinical and subjects with type 1 diabetes against neo- (C-peptide-IAPP1, C-peptide-IAPP2 and C-peptide-NPY) and native epitopes (C13–32, C19-A3, C22-A5). The dashed line represents the cut-off for positivity for the stimulation index (three for interferon-γ and IL-10 and two for IL-17). (B) Prevalence of IFN-γ (red) IL-10 (blue) and IL-17 (purple) responses measured by ELISPOT in preclinical and subjects with type 1 diabetes against individual peptides of neo- (open bars) and native (filled bars) epitopes.

Figure 2

Cytokine T cell responses were measured by ELISPOT, and data are expressed as SI [stimulation index (mean spot number of test peptide/mean spot number of diluent)]. (A) Magnitude of responses as depicted by SI for IFN-γ (red) IL-10 (blue) and IL-17 (purple) responses against pooled neo- and native epitopes in preclinical and subjects with type 1 diabetes. (B) Prevalence of IFN-γ (red) IL-10 (blue) and IL-17 (purple) responses in preclinical and subjects with type 1 diabetes against pooled peptides of neo- (open bars) and native (filled bars) epitopes.

Figure 3

Cytokine T cell responses were measured by ELISPOT, and data are expressed as SI [stimulation index (mean spot number of test peptide/mean spot number of diluent)]. (A) Magnitude of IFN-γ (red) IL-10 (blue) and IL-17 (purple) responses depicted as stimulation indices for individual peptides of neoepitopes (three on the left) and native (three on the right) in preclinical and subjects with type 1 diabetes. (B) Frequency of IFN-γ (red) IL-10 (blue) and IL-17 (purple) responses to individual peptides of neoepitopes (three on the left) and native (three on the right) in preclinical and subjects with type 1 diabetes.

Figure 4

PBMCs from subjects ND01 and ND02 were stimulated with native (proinsulin peptides: C13–32, C19-A3, C22-A5; GAD peptides: 115–125, 265–284) and neoepitopes [C-pep-IAPP1, C-pep-NPY, GAD 115–127 (120E), GAD 265–284 (272 cit)] and each patient/pool was labeled with a different hashtag as described in Methods. Antigen-responsive cells were identified by expression of CD154/CD69, and immune profiling was subsequently conducted on single cells. Cells from subjects (A) ND01 and ND02 separate into (B) three clusters (0–3) based on gene expression similarity. (C) Clusters differ by expression of memory and naïve cell specific genes. (D) Cells reacting to native and neoepitopes are similarly split between clusters and (E) have similar patterns of expression.

Figure 5

PBMCs from subjects ND01 and ND02 were stimulated with native [proinsulin peptides: C13–32, C19-A3, C22-A5; GAD peptides: 115–125, 265–284) and neoepitopes (C-pep-IAPP1, C-pep- NPY, GAD 115-127 (120E), GAD 265-284 (272 cit)]. Antigen-responsive cells were identified by expression of CD154/CD69, and immune profiling was conducted on single cells. (A) Cells were stimulated with native (right) and neoepitopes (left), and gene expression (depicted in pink) of (A) IL-17A, IL-22, IL-32, TNF-α, and IFN-γ and (B) IL-4 and IL-10 was examined. Level of expression corresponds to greater intensity of color.

Figure 6

PBMCs from subjects with type 1 diabetes were stimulated with native [proinsulin peptides: C13–32, C19-A3, C22-A5; GAD peptides: 115–25, 265–284) and neoepitopes (C-pep-IAPP1, C-pep-NPY, GAD 115–127 (120E), GAD 265–284 (272 cit)]. Antigen-responsive cells were identified by and sorted on expression of CD154/CD69. Immune profiling was conducted on single cells from which cDNA was synthesized, followed by library construction and sequencing as described in Methods. TCR clonotypes (both α and β chains) were examined in patients ND01 (top panel) and ND02 (bottom panel). T cells responding to native epitopes are shown in red and those responding to neoepitopes are shown in blue. Clusters are marked by point shapes (circle, triangle, and square).