Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors

Abstract

The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8_186-194 (ZnT8_186-194) and other islet epitopes elicit interferon-γ secretion by CD8⁺ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8_186-194-reactive CD8⁺ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8⁺ T cells reactive to ZnT8_186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8_186-194-reactive CD8⁺ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8_186-194-reactive CD8⁺ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8⁺ T cells. In contrast, ZnT8_186-194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8⁺ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Fig. 1. ZnT8_186–194-reactive CD8⁺ T-cell clones from patient D222D

(A) Frozen-thawed PBMCs were cultured with ZnT8_186–194 or no peptide and stained with PE/APC-labeled ZnT8_186–194 MMrs. (B) ZnT8_186–194 and control MMr stains for one clone obtained from single-sorted ZnT8_186–194/ZnT8_186–194 double-MMr⁺ cells. (C) Percent intracellular TNF-α⁺ D222D clone 1 cells stimulated for 6 h with K562-A2 cells pulsed with ZnT8_186–194 or Flu MP_58–66 peptide. (D) Percent lysis of FarRed-labeled LCL targets pulsed with ZnT8_186–194 (top) or Flu MP_58–66 peptide (bottom) and cultured for 24 h with CFSE-labeled D222D clone 3 at increasing E/T ratios. (E) Percent lysis of LCL targets cultured with D222D clones 1, 2 or 3 (mean±SEM; each clone is depicted in Fig. S1D–F). (F) Lysis of cognate peptide-pulsed LCLs cultured for 4 h with D222D clone 2 or a MelanA_26–35-reactive clone (E/T 1/1) in the presence of concanamycin A (CMA), brefeldin A (BFA), CMA and BFA, anti-FasL or control IgG1. *p=0.015, **p=0.009, ***p<0.001 by Student’s t test. Results are mean±SEM of triplicate measurements from one of three experiments. (G) Percent surface CD107a⁺ D222D clone 1 cells stimulated as in (C). For panels A, C, G, gate is on viable CD8⁺ cells.

Fig. 2. Ag avidity, Ag sensitivity and polyfunctionality of ZnT8_186–194-reactive CD8⁺ T-cell clones

(A) ZnT8_186–194 MMr staining in the absence (light grey) or presence (dark grey) of dasatinib. The dotted profile indicates the unstained control. (B) ZnT8_186–194 MMr median fluorescence intensity (MFI) for the indicated clones in the absence (left) or presence (right) of dasatinib. Bars indicate median values. Results are representative of two separate experiments. (C) The indicated clones were stimulated for 6 h with ZnT8_186–194-pulsed K562-A2 cells and percent cytokine⁺ cells out of viable CD8⁺ cells calculated. Results are representative of three independent experiments. (D–E) Half maximal effective peptide concentration (EC50; D) and maximal cytokine response (percent cytokine⁺ cells at optimal peptide concentrations; E) for clones stimulated as above. Bars indicate median values. Results are representative of two to four separate experiments. *p=0.014 by Mann-Whitney test. (F) Polyfunctionality distribution of T1D (left) and healthy clones (right). Percent T cells producing 0 to 4 cytokines among TNF-α, IFN-γ, IL-2 and MIP-1β upon exposure to ZnT8_186–194-pulsed K562-A2 cells (100 µM) are shown.

Fig. 3. Target cell lysis by ZnT8_186–194-reactive CD8⁺ T-cell clones

(A–C) Lysis of K562-A2 cells transfected (open triangles) or not (open circles) with a full-length ZnT8 plasmid and cultured for 24 h with clones D222D 2 (A), H017N A1 (B) or H314C 6C4 (C). Filled symbols indicate ZnT8_186–194-pulsed target cells (10 µM). Results are presented as mean±SEM of triplicate wells from two separate experiments. (D–G) Real-time cytotoxicity for the indicated clones vs. HLA-A2⁺ ECN90 (white triangles) or control HLA-A2⁻ EndoC-βH2 β-cell targets (white circles) (E/T 2/1). Black and grey symbols indicate the corresponding targets pulsed with 10 µM ZnT8_186–194 or GAD_114–122 peptide, respectively. Mean±SEM of triplicate measurements are shown at each time point. Results are representative of at least two separate experiments. (H) Percent maximal HLA-A2⁺ ECN90 and HLA-A2⁻ EndoC-βH2 β-cell lysis by the indicated clones (T1D, grey symbols; healthy, white symbols; control, horizontal dotted line) in the absence or presence of ZnT8_186–194 peptide. Bars indicate median values. Lysis was calculated from the cytotoxicity profiles as in panels D–G.

Fig. 4. In-silico search for CDR3β aminoacid sequences from ZnT8_186–194-reactive CD8⁺ T-cell clones

(A–C) Prevalence of the CDR3β aminoacid sequences from clones D010R 1E2 (A), H328C 8E8 (B) and H034O 141B9 (C) among HLA-A2⁺ T1D (n=5), aAb⁺ (n=5) and healthy subjects (n=10), as assessed by in-silico analysis of TCRβ repertoires obtained from the indicated CD8⁺ and CD4⁺ T-cell subsets. (D) In-silico search for the same CDR3β aminoacid sequences in the repertoire of CD8⁺, conventional CD4⁺ (Tconv; CD127⁺) and regulatory CD4⁺ (Treg; CD25⁺CD127⁻) T cells obtained from nPOD PLN, spleen and inguinal lymph node (ILN) samples via the online database http://clonesearch.jdrfnpod.org. For each cell type and tissue, the first, second and third columns refer to clones D010R 1E2, H034O 141B9 and H328C 8E8, respectively. Dark and light grey cells indicate negative and positive samples, respectively. Frequencies per 10⁶ TCRs are annotated, and underlining indicates samples with a nucleotide sequence match. White cells indicate unavailable samples. Pancreatic NET, neuro-endocrine tumor.

Fig. 5. Ex-vivo frequencies and Ag-experienced phenotypes of circulating islet-reactive CD8⁺ T cells

(A) ZnT8_186–194, MelanA_26–35 and Flu MP_58–66 MMr⁺CD8⁺ cells were stained ex-vivo and counted (see Fig. S7). Frequencies out of total CD8⁺ T cells are depicted for T1D adults (red circles), T1D children (crossed red circles), age/sex-matched healthy adults (blue circles) and children (crossed blue circles). *p≤0.05, **p=0.002, ***p≤0.0003. (B) Percent Ag-experienced cells out of total MMr⁺ cells. *p≤0.03, **p=0.004, ***p=0.0007. (C) Absolute frequencies of the corresponding Ag-experienced fractions. *p≤0.03, **p≤0.01, ***p≤0.0001. (D) MMr⁺CD8⁺ cells reactive to the indicated islet epitopes were stained ex-vivo and counted (see Fig. S10A). Frequencies out of total CD8⁺ T cells are depicted as in panel A. *p=0.02. (E) Percent Ag-experienced cells out of total MMr⁺ cells. (F) Absolute frequencies of the corresponding Ag-experienced fractions. Bars display median values. The median number of MMr⁺ events and total CD8⁺ T cells analyzed are indicated for each distribution. Significance was determined using the Mann-Whitney test. For panels A and D, data-points with <300,000 CD8⁺ T cells and <5 MMr⁺ cells were excluded. For panels B–C and E–F, data-points with <5 MMr⁺ cells were excluded.

Fig. 6. SLC30A8 and INS gene expression in mTECs and circulating islet-reactive CD8⁺ T-cell frequencies in HLA-A2⁺ and HLA-A2⁻ healthy donors

(A) SLC30A8 RT-PCR strategy. Forward primers spanned exons 5 to 8, reverse primers spanned either exon 11 or the 3’-UTR. The position of the ZnT8_186–194-coding region is shown. (B) SLC30A8 expression in mTECs, using the indicated forward primers and the exon 11 reverse primer. (C) SLC30A8 expression in mTECs from donor #64 (previously testing positive with the exon 8 forward primer) and #211 (previously testing negative), using the 3’-UTR reverse primer. (D) INS RT-PCR strategy. Forward primers spanned both or neither of the PPI_6–14 and PPI_15–24 regions, reverse primers spanned either the 3’-UTR or exon 2 (PCR products 1 and 2, respectively). (E) INS expression in thymuses pooled from 5–8 donors. (F) Ex-vivo MMr⁺CD8⁺ cell frequencies in age/sex-matched, EboV- and HCV-seronegative HLA-A2⁺ and HLA-A2⁻ healthy donors. (G) Percent Ag-experienced MMr⁺ cells. Bars indicate median values. The median number of MMr⁺ events is indicated, with a median of 1×10⁶ total CD8⁺ T cells analyzed. *p≤0.03, **p=0.008, ***p≤0.0004 by Mann-Whitney test. For panels G, data points with <5 MMr⁺ cells were excluded. NA, not available.

Fig. 7. ZnT8_186–194-reactive CD8⁺ T cells cross-recognize a B. stercoris mimotope

(A–C) Four donors with sizable ZnT8_186–194 MMr⁺CD8⁺ T-cell fractions were selected. A first PBMC aliquot received PE/BV786-labeled ZnT8_186–194 MMrs and PE/BV711-labeled EboV NP_202–210 MMrs. For the second aliquot, PE-labeled B. stercoris MMrs replaced the PE-labeled ZnT8_186–194 MMrs. (A) Overlay of ZnT8_186–194/ZnT8_186–194 MMr⁺ (blue) and ZnT8_186–194/B. stercoris MMr⁺ cells (red). (B) Negative control staining of ZnT8_186–194/EboV NP_202–210 MMr⁺ cells. (C) Positive control staining of EboV NP_202–210/EboV NP_202–210 MMr⁺ cells from the first and second aliquot. The frequencies of MMr⁺ out of total CD8⁺ T cells are indicated. (D) Four ZnT8_186–194-reactive CD8⁺ T-cell clones (D222D 2, D349D 178B9, H017N A1, H328C 9C8) were stained with BV786/PE-labeled ZnT8_186–194, PE-labeled B. stercoris and BV650-labeled MelanA_26–35 MMrs. The ZnT8_186–194/B. stercoris cross-reactive clone H017N is shown, from left to right: ZnT8_186–194/B. stercoris MMr⁺; ZnT8_186–194/ZnT8_186–194 MMr⁺; and negative control ZnT8_186–194/MelanA_26–35 and B. stercoris/MelanA_26–35 MMr⁺ cells. (E) The H017N clone was stimulated with peptide-pulsed LCLs (0.1 µM, 6 h). Percent cytokine⁺ cells are shown as mean±SEM of two experiments. p=0.008 for Wilcoxon signed-rank comparison of pooled cytokine responses between B. stercoris and ZnT8_186–194, MelanA_26–35 or no peptide, and between ZnT8_186–194 and MelanA_26–35 or no peptide.

Fig. 8. In-situ ZnT8_186–194 MMr staining of nPOD pancreas and PLN sections

(A–E) Representative pancreas images (20X magnification; scale bar 100 µm) from cases T1D #6161 (A), aAb⁺ #6347 (B) and non-diabetic (ND) #6289 (C). Red arrows indicate MMr⁺ cells and the dotted areas of panels A–B are magnified in panels D (scale bar 80 µm) and E (scale bar 50 µm), respectively. (F) Consecutive sections from ZnT8_186–194 MMr⁺ pancreata were probed with negative control MelanA_26–35 MMrs. A representative image from T1D case #6211 is shown on the left, and a positive control staining on skin sections from a vitiligo patient is shown on the right (20X; scale bar 100 µm). (G) Representative PLN image (20X; scale bar 100 µm) from T1D case #6161. (H) Magnification of the dotted area of panel G (scale bar 40 µm). (I–J) Number of ZnT8_186–194 and MelanA_26–35 MMr⁺ cells/mm² section area of pancreas (I) and PLNs (J). Each point represents an individual case (detailed in Table S5). Bars indicate median values. *p≤0.05, **p≤0.009 by Mann-Whitney test. NA, not assessed.