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

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Science Immunology  02 Feb 2018:
Vol. 3, Issue 20, eaao4013
DOI: 10.1126/sciimmunol.aao4013
  • Fig. 1 ZnT8186–194-reactive CD8+ T cell clones from patient D222D.

    (A) Frozen-thawed PBMCs were cultured with ZnT8186–194 or no peptide and stained with phycoerythrin (PE)/allophycocyanin (APC)–labeled ZnT8186–194 MMrs. (B) ZnT8186–194 and control MMr stains for one clone obtained from single-sorted ZnT8186–194/ZnT8186–194 double-MMr+ cells. (C) Percentage of intracellular TNF-α+ D222D clone 1 cells stimulated for 6 hours with K562-A2 cells pulsed with ZnT8186–194 or Flu MP58–66 peptide. (D) Percent lysis of Far Red–labeled LCL targets pulsed with ZnT8186–194 (top) or Flu MP58–66 peptide (bottom) and cultured for 24 hours with CFSE-labeled D222D clone 3 at increasing E/T ratios. (E) Percent lysis of LCL targets cultured with D222D clone 1, 2, or 3 (mean ± SEM; each clone is depicted in fig. S1, D to F). (F) Lysis of cognate peptide–pulsed LCLs cultured for 4 hours with D222D clone 2 or a MelanA26–35-reactive clone (E/T, 1:1) in the presence of concanamycin A (CMA), brefeldin A (BFA), CMA and BFA, anti-FasL (aFasL), or control IgG1 (immunoglobulin G1). *P = 0.015, **P = 0.009, and ***P < 0.001 by Student’s t test. Results are mean ± SEM of triplicate measurements from one of three experiments. (G) Percentage of surface CD107a+ D222D clone 1 cells stimulated as in (C). For (A), (C), and (G), the gate is on viable CD8+ cells.

  • Fig. 2 Ag avidity, Ag sensitivity, and polyfunctionality of ZnT8186–194-reactive CD8+ T cell clones.

    (A) ZnT8186–194 MMr staining in the absence (light gray) or presence (dark gray) of dasatinib. The dotted profile indicates the unstained control. (B) ZnT8186–194 MMr 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 hours with ZnT8186–194-pulsed K562-A2 cells, and the percentage of cytokine+ cells out of viable CD8+ cells was calculated. Results are representative of three independent experiments. (D and E) 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). Percentage of T cells producing zero to four cytokines among TNF-α, IFN-γ, IL-2, and MIP-1β upon exposure to ZnT8186–194-pulsed K562-A2 cells (100 μM) are shown.

  • Fig. 3 Target cell lysis by ZnT8186–194-reactive CD8+ T cell clones.

    (A to C) Lysis of K562-A2 cells transfected (open triangles) or not (open circles) with a full-length ZnT8 plasmid and cultured for 24 hours with clones D222D 2 (A), H017N A1 (B), or H314C 6C4 (C). Filled symbols indicate ZnT8186–194-pulsed target cells (10 μM). Results are presented as mean ± SEM of triplicate wells from two separate experiments. (D to G) Real-time cytotoxicity for the indicated clones versus HLA-A2+ ECN90 (white triangles) or control HLA-A2 EndoC-βH2 β cell targets (white circles) (E/T, 2:1). Black and gray symbols indicate the corresponding targets pulsed with 10 μM ZnT8186–194 or GAD114–122 peptide, respectively [ZnT8186–194 or MelanA26–35 for the H004N clone MelanA in panel (G)]. Means ± 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, gray symbols; healthy, white symbols; control H004N clone MelanA, horizontal dotted line) in the absence or presence of the ZnT8186–194 peptide. Bars indicate median values. Lysis was calculated from the cytotoxicity profiles as in (D) to (G).

  • Fig. 4 In silico search for CDR3β amino acid sequences from ZnT8186–194-reactive CD8+ T cell clones.

    (A to C) Prevalence of the CDR3β amino acid sequences from clones D010R 1E2 (A), H328C 8E8 (B), and H034O 141B9 (C) among HLA-A2+ T1D (n = 5), aAb+ (n = 5), and healthy individuals (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 amino acid 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 gray 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, neuroendocrine tumor.

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

    (A) ZnT8186–194, MelanA26–35, and Flu MP58–66 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- and sex-matched healthy adults (blue circles), and children (crossed blue circles). *P ≤ 0.05, **P = 0.002, and ***P ≤ 0.0003. (B) Percentage of Ag-experienced cells out of total MMr+ cells. *P ≤ 0.03, **P = 0.004, and ***P = 0.0007. (C) Absolute frequencies of the corresponding Ag-experienced fractions. *P ≤ 0.03, **P ≤ 0.01, and ***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 (A). *P = 0.02. (E) Percentage of 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 (A) and (D), data points with <300,000 CD8+ T cells and <5 MMr+ cells were excluded. For (B), (C), (E), and (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, and reverse primers spanned either exon 11 or the 3′UTR. The position of the ZnT8186–194-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. bp, base pair. (D) INS RT-PCR strategy. Forward primers spanned both or neither of the PPI6–14 and PPI15–24 regions, and reverse primers spanned either the 3′UTR or exon 2 (PCR products 1 and 2, respectively). (E) INS expression in thymuses pooled from five to eight donors. (F) Ex vivo MMr+CD8+ cell frequencies in age- and 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, and ***P ≤ 0.0004 by Mann-Whitney test. For (G), data points with <5 MMr+ cells were excluded. NA, not available.

  • Fig. 7 ZnT8186–194-reactive CD8+ T cells cross-recognize a B. stercoris mimotope.

    (A to C) Four donors with sizable ZnT8186–194 MMr+CD8+ T cell fractions were selected. A first PBMC aliquot received PE/BV786-labeled ZnT8186–194 MMrs and PE/BV711-labeled EboV NP202–210 MMrs. For the second aliquot, PE-labeled B. stercoris MMrs replaced the PE-labeled ZnT8186–194 MMrs. (A) Overlay of ZnT8186–194/ZnT8186–194 MMr+ (blue) and ZnT8186–194/B. stercoris MMr+ cells (red). (B) Negative control staining of ZnT8186–194/EboV NP202–210 MMr+ cells. (C) Positive control staining of EboV NP202–210/EboV NP202–210 MMr+ cells from the first and second aliquot. The frequencies of MMr+ out of total CD8+ T cells are indicated. (D) Four ZnT8186–194-reactive CD8+ T cell clones (D222D 2, D349D 178B9, H017N A1, and H328C 9C8) were stained with BV786/PE-labeled ZnT8186–194, PE-labeled B. stercoris, and BV650-labeled MelanA26–35 MMrs. The ZnT8186–194/B. stercoris cross-reactive clone H017N is shown, from left to right: ZnT8186–194/B. stercoris MMr+, ZnT8186–194/ZnT8186–194 MMr+, negative control ZnT8186–194/MelanA26–35, and B. stercoris/MelanA26–35 MMr+ cells. (E) The H017N clone was stimulated with peptide-pulsed LCLs (0.1 μM; 6 hours). Percentages of 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 ZnT8186–194, MelanA26–35, or no peptide and between ZnT8186–194 and MelanA26–35 or no peptide.

  • Fig. 8 In situ ZnT8186–194 MMr staining of nPOD pancreas and PLN sections.

    (A to E) Representative pancreas images from cases T1D #6161 (A), aAb+ #6347 (B), and nondiabetic (ND) #6289 (C) (magnification, ×20; scale bars, 100 μm). Red arrows indicate MMr+ cells, and the dotted areas of (A) and (B) are magnified in (D) (scale bar, 80 μm) and (E) (scale bar, 50 μm), respectively. (F) Consecutive sections from ZnT8186–194 MMr+ pancreata were probed with negative control MelanA26–35 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 (magnification, ×20; scale bars, 100 μm). (G) Representative PLN image (magnification, ×20; scale bar, 100 μm) from T1D case #6161. (H) Magnification of the dotted area of (G) (scale bar, 40 μm). (I and J) Number of ZnT8186–194 and MelanA26–35 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 and **P ≤ 0.009 by Mann-Whitney test. NA, not assessed.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/20/eaao4013/DC1

    Fig. S1. Cytokine secretion and cytotoxicity of ZnT8186–194-reactive CD8+ T cells from T1D patient D222D.

    Fig. S2. CD8+ T cell recognition and HLA-A2 binding of ZnT8186–194 and ZnT8185–194 epitope variants.

    Fig. S3. Ag sensitivity correlates with Ag avidity and polyfunctionality in ZnT8186–194-reactive CD8+ T cell clones.

    Fig. S4. Modulation of HLA class I and ZnT8 expression in human β cell lines.

    Fig. S5. TCR sequences of ZnT8186–194-reactive CD8+ T cell clones.

    Fig. S6. ZnT8186–194-reactive clonotype-specific TaqMan assays.

    Fig. S7. Gating strategy for the analysis of ZnT8186–194, MelanA26–35, and Flu MP58–66 MMr+CD8+ T cells.

    Fig. S8. IFN-γ secretion by ZnT8186–194-reactive CD8+ T cells.

    Fig. S9. Gene expression in ex vivo single-sorted ZnT8186–194 MMr+CD8+ T cells.

    Fig. S10. Extended combinatorial MMr panel for the analysis of multiple islet-reactive CD8+ T cell populations, and reproducibility of ex vivo MMr assays.

    Fig. S11. CD27, CD28, and CD95 expression on ZnT8186–194-reactive CD8+ T cells.

    Fig. S12. Representative MMr and CD45RA/CCR7 dot plots for HLA-A2+ and HLA-A2 healthy donors depicted in Fig. 6 (F and G).

    Fig. S13. Correlation between the frequency of MMr+CD8+ T cells and the Ag-experienced fraction within the same MMr+CD8+ population.

    Table S1. Summary of ZnT8186–194-reactive CD8+ T cell clones.

    Table S2. Characteristics of study patients for in silico TRB analyses.

    Table S3. Characteristics of HLA-A2+ study patients for ex vivo MMr studies.

    Table S4. Characteristics of HLA-A2+ and HLA-A2 healthy donors for ex vivo MMr studies.

    Table S5. Characteristics of nPOD cases for in situ ZnT8186–194 MMr staining.

    Table S6. Primers used for gene expression analysis of the individual ZnT8186–194 MMr+CD8+ T cells depicted in fig. S9A.

    Members of the ImMaDiab Study Group

    Data file S1. Raw data from figure graphs (Excel).

    Reference (43)

  • Supplementary Materials

    Supplementary Material for:

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

    Slobodan Culina, Ana Ines Lalanne, Georgia Afonso, Karen Cerosaletti, Sheena Pinto, Guido Sebastiani, Klaudia Kuranda, Laura Nigi, Anne Eugster, Thomas Østerbye, Alicia Maugein, James E. McLaren, Kristin Ladell, Etienne Larger, Jean-Paul Beressi, Anna Lissina, Victor Appay, Howard W. Davidson, Søren Buus, David A. Price, Matthias Kuhn, Ezio Bonifacio, Manuela Battaglia, Sophie Caillat-Zucman, Francesco Dotta, Raphael Scharfmann, Bruno Kyewski, Roberto Mallone,* ImMaDiab Study Group

    *Corresponding author. Email: roberto.mallone{at}inserm.fr

    Published 2 February 2018, Sci. Immunol. 3, eaao4013 (2017)
    DOI: 10.1126/sciimmunol.aao4013

    This PDF file includes:

    • Fig. S1. Cytokine secretion and cytotoxicity of ZnT8186?194-reactive CD8+ T cells from T1D patient D222D.
    • Fig. S2. CD8+ T cell recognition and HLA-A2 binding of ZnT8186?194 and ZnT8185?194 epitope variants.
    • Fig. S3. Ag sensitivity correlates with Ag avidity and polyfunctionality in ZnT8186?194-reactive CD8+ T cell clones.
    • Fig. S4. Modulation of HLA class I and ZnT8 expression in human β cell lines.
    • Fig. S5. TCR sequences of ZnT8186?194-reactive CD8+ T cell clones.
    • Fig. S6. ZnT8186?194-reactive clonotype-specific TaqMan assays.
    • Fig. S7. Gating strategy for the analysis of ZnT8186?194, MelanA26?35, and Flu MP58?66 MMr+CD8+ T cells.
    • Fig. S8. IFN-γ secretion by ZnT8186?194-reactive CD8+ T cells.
    • Fig. S9. Gene expression in ex vivo single-sorted ZnT8186?194 MMr+CD8+ T cells.
    • Fig. S10. Extended combinatorial MMr panel for the analysis of multiple isletreactive CD8+ T cell populations, and reproducibility of ex vivo MMr assays.
    • Fig. S11. CD27, CD28, and CD95 expression on ZnT8186?194-reactive CD8+ T cells.
    • Fig. S12. Representative MMr and CD45RA/CCR7 dot plots for HLA-A2+ and HLA-A2 healthy donors depicted in Fig. 6 (F and G).
    • Fig. S13. Correlation between the frequency of MMr+CD8+ T cells and the Agexperienced fraction within the same MMr+CD8+ population.
    • Table S1. Summary of ZnT8186?194-reactive CD8+ T cell clones.
    • Table S2. Characteristics of study patients for in silico TRB analyses.
    • Table S3. Characteristics of HLA-A2+ study patients for ex vivo MMr studies.
    • Table S4. Characteristics of HLA-A2+ and HLA-A2 healthy donors for ex vivo MMr studies.
    • Table S5. Characteristics of nPOD cases for in situ ZnT8186?194 MMr staining.
    • Table S6. Primers used for gene expression analysis of the individual ZnT8186?194 MMr+CD8+ T cells depicted in fig. S9A.
    • Members of the ImMaDiab Study Group
    • Reference (43)

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