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Suppression of diabetes by accumulation of non–islet-specific CD8+ effector T cells in pancreatic islets

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Science Immunology  23 Mar 2018:
Vol. 3, Issue 21, eaam6533
DOI: 10.1126/sciimmunol.aam6533
  • Fig. 1 Ag governs CTL behavior at islets but is not required for their accumulation in large numbers.

    (A) Islet (dashed line) in the pancreas of a diabetic RIP-GP mouse where islet-specific P14 CTLs (red) and non–islet-specific OT-I CTLs (green) have gathered. (B) An islet (dashed line) in the pancreas of a wt C57Bl/6 mouse immunized in the same way as in (A) displaying a few CTLs in the pancreatic parenchyma. (C and D) The levels of T cell proliferation were similar between the two strains of mice, as judged by T cell content in spleens. (E to G) The densities of accumulated CTLs of both specificities (P14 and OT-I) were higher in the islets than in the surrounding pancreatic parenchyma, as quantified in (E) (n = 5 mice) and visualized in (F) and (G), where the yellow line across a part of the pancreas in (F) is represented in the histograms over fluorescent signals in (G). (H) Islets in mouse pancreata were imaged in anesthetized mice using a vacuum imaging window where the pancreas was immobilized, enabling long-term imaging. (I and J) A still frame (z-projection) (I) from an intravital recording of an inflamed islet in a RIP-GP mouse (red P14 CD8+ T cells and green OT-I CD8+ T cells) and the resulting T cell tracks (J). (B) and (C) correspond to movie S3. (K and L) OT-I CTLs consistently displayed higher migration speeds than the islet-specific P14 CTLs (K) and were kept, on average, further away from the islet center (L). Data in (J) to (L) are from one of six representative experiments. Data are means ± SEM. *P < 0.05, two-tailed unpaired Mann-Whitney U tests. Scale bars, 20 μm (A to D), 30 μm (F), and 50 μm (I and J).

  • Fig. 2 Abundance of non–islet-specific CTLs at islets is immune-suppressive.

    (A and C) Adoptive transfer of equal amounts (1 × 106) of P14 and OT-I CD8+ T cells resulted in an abundance of non–islet-specific OT-I CTLs at islets on day 9 in RIP-GP mice (A) compared with when the ratio was changed to 1000:1 (P14:OT-I) (C). (B and D) Follow-up studies of the mice on day 16 showed that the infiltrate was gone, and in the HI (1:1 ratio) group, the islets were found to be mostly intact (B), whereas in the LO (1000:1 ratio) group, very few β cells were found (D). Scale bars, 30 μm. (E) A majority of the HI group (six of eight) displayed normoglycemia, whereas only one of eight was normoglycemic in the LO group (*P = 0.0058, 1:1 versus 1:1000, log-rank test). Different ratios of cell transfers suggested a dose-dependent mechanism because 50% of the mice in the group receiving a 10:1 ratio remained normoglycemic. (F to H) These scenarios were repeated in three different transgenic models that express model Ags on β cells: RIP-GP (six of eight were normoglycemic, representative of four independent experiments) (F), RIP-mOVA (five of six were normoglycemic, representative of two independent experiments) (G), and RIP-OVAlow (three of four were normoglycemic, representative of two independent experiments) (H).

  • Fig. 3 Protection from autoimmunity is not due to the expansion of Treg subsets.

    (A to D) Flow plots showing similar representation of CD4+ T cells at islets in the two scenarios (A and B) but different amounts of CD8+ T cells (C and D). (E) Despite having lower amounts of total CD8+ T cells, LO mice had more islet-specific P14 T cells in the pancreas. (F and G) The distributions of Ag specificity of the CD8+ T cells present in the pancreas are visualized in (F) and (G), where each dot represents 1of 100. (H and I) Representative fixed, frozen tissue sections from HI and LO pancreata displaying the TCR-tg cells [red, P14 (islet-specific); green, OT-I (non–islet-specific)] and endogenous CD8+ T cells [blue, anti-CD8 monoclonal antibody (mAb) (polyclonal)] in islets (dashed lines). Scale bar, 20 μm. (J to L) CD4+/CD25+/FoxP3+ Treg fractions were not different between the two scenarios. (M and N) The abundant population of non–islet-specific T cells in the HI scenario had not transformed into a CD8+ Treg type as judged by the expression of FoxP3 (M) and CD122 (N). Groups are representative of at least three independent experiments. Data are means ± SEM. *P < 0.05, two-tailed unpaired Mann-Whitney U tests. MFI, mean fluorescence intensity.

  • Fig. 4 High numbers of non–islet-specific CTLs attenuate effector functions of islet-specific CTLs.

    (A) Flow cytometric analysis of a range of surface markers on islet-specific P14 CD8+ T cells visualized as a heat map [based on arbitrary intensity units (MFI)]. Significant differences were found in CD69, KLRG1, and PD-1. (B and C) The expression of the activation marker CD69 on Ag-specific P14 CTLs was higher in the group receiving low amounts of non–islet-specific CD8+ T cells. (D and E) The exhaustion marker PD-1 was found to be highly expressed in the HI scenario (D), quantified in (E). Results are representative of two to three independent experiments. Data are means ± SEM. *P < 0.05, two-tailed unpaired Mann-Whitney U tests. (F to J) Islet-specific P14 CTLs isolated from pancreatic draining lymph nodes were stimulated in vitro with their cognate Ag GP33–41 and revealed less effector cytokine production (IFN-γ) [(F) and (G)] and increased IL-10 production [(H) and (I)] in mice receiving HI amounts of non–islet-specific TCR-tg CD8+ T cells; this is quantified in (J). *P < 0.05, two-tailed unpaired Mann-Whitney U tests. FSC, forward scatter.

  • Fig. 5 Low accessibility to Ag for CTLs leads to low proliferation of effectors.

    Intravital observations in the pancreas of RIP-GPxCX3CR1+/GFP mice from the HI and LO scenarios revealed less interactions between islet-specific P14 CTLs and GFP+ APCs in the HI scenario compared with the LO scenario. (A and B) Still frames from representative recordings for HI (A) (movie S4) and for LO (B) (movie S5). (C) Interactions are quantified as the arrest coefficient (fraction of time spent in a nonmoving state). Data are representative of groups of six mice per treatment. (D and E) APC occupancy by islet-specific CTLs and non–islet-specific CTLs was further analyzed in paraformaldehyde-fixed, frozen pancreas sections from RIP-GPxCX3CR1+/GFP mice receiving HI (D) or LO (E) amounts of non–islet-specific CD8+ T cells (n = 4 mice per group). Bars next to images display fractional occupancy of APCs by the indicated cell type. (F to H) The difference in APC occupancy could not be explained by changes in islet APC subsets, as shown in (F) for macrophages, in (G) for migratory DCs, and in (H) for plasmacytoid DCs (n = 3 to 4 mice per group). (I and J) The low level of APC interactions in the HI scenario likely led to a lower level of proliferation of effectors, as assessed by transfer of CellTrace Violet–stained Ly5.1+ P14 CD8+ T cells. n = 5 mice per group. Results are representative of two independent experiments. Scale bars, 10 μm. Data are means ± SEM. *P < 0.05, two-tailed unpaired Mann-Whitney U tests.

  • Fig. 6 Non–viral Ag–specific CTLs dampen inflammation in a mouse model of viral meningitis.

    (A) Mice were inoculated intracerebrally with LCMV. One group (black line) had been infected with LCMV intraperitoneally 1 month before intracerebral infection and mounted a memory response, and mice were thereby protected. Mice receiving intracerebral LCMV without previous induced memory succumbed to the infection 7.25 days (median) after inoculation (red line). In cell transfer experiments similar to the HI and LO scenarios in the T1D models, 1 × 103 or 1 × 106 non–LCMV-specific OT-I CD8+ T cells were transferred and activated in vivo through peptide immunization. High amounts of OT-I led to a significant delay (P = 0.0006, 1 × 103 OT-I versus 1 × 106 OT-I, log-rank test) in time to death (n = 6 mice per group; data are representative of two independent experiments). Brains from the mice were examined 7 days after intracerebral infection. (B and C) No morphological differences or major differences in immune infiltrates could be detected between the groups receiving 1 × 106 (B) or 1 × 103 (C) OT-I CD8+ T cells (n = 4 mice per group). Scale bars, 1 mm. (D and E) Micrographs of periventricular areas in (D) and (E) represent the squares in (B) and (C) and have been stained using anti-CD8 mAb (gray), Hoechst for nuclei (blue), and OT-I cells expressing GFP (green). Scale bars, 20 μm. Bars next to the micrographs represent the clonal distribution of CD8+ T cells. (F) Overall numbers of CD8+ T cell clones per field of view in periventricular regions of brains from mice receiving transfers of 1 × 106 (HI) or 1 × 103 (LO) OT-I CD8+ T cells. *P < 0.05, two-way ANOVA.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/21/eaam6533/DC1

    Methods

    Fig. S1. Ag-driven mouse models of T1D.

    Fig. S2. CTL activation and expansion is a requirement for pancreatic recruitment.

    Fig. S3. Immune suppression by non–Ag-specific CD8+ T cells was not due to a “transfer bias.”

    Fig. S4. No signs of differences in the cytokine environment in the pancreas.

    Movie S1. Activated CTLs infiltrate islets irrespective of the presence of their cognate Ag.

    Movie S2. No islet infiltration in the absence of cognate Ag and islet inflammation.

    Movie S3. Differential behavior of islet-specific CTLs and non–islet-specific CTLs at islets.

    Movie S4. High amounts of CTLs not recognizing local Ag reduces the number of interactions with APCs.

    Movie S5. High amounts of CTLs not recognizing local Ag reduces the number of interactions with APCs.

    Reference (48)

  • Supplementary Materials

    Supplementary Material for:

    Suppression of diabetes by accumulation of non–islet-specific CD8+ effector T cells in pancreatic islets

    Gustaf Christoffersson, Grzegorz Chodaczek, Sowbarnika S. Ratliff, Ken Coppieters, Matthias G. von Herrath*

    *Corresponding author. Email: matthias{at}lji.org

    Published 23 March 2018, Sci. Immunol. 3, eaam6533 (2018)
    DOI: 10.1126/sciimmunol.aam6533

    This PDF file includes:

    • Methods
    • Fig. S1. Ag-driven mouse models of T1D.
    • Fig. S2. CTL activation and expansion is a requirement for pancreatic recruitment.
    • Fig. S3. Immune suppression by non–Ag-specific CD8+ T cells was not due to a ?transfer bias.?
    • Fig. S4. No signs of differences in the cytokine environment in the pancreas.
    • Reference (48)

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Activated CTLs infiltrate islets irrespective of the presence of their cognate Ag.
    • Movie S2 (.avi format). No islet infiltration in the absence of cognate Ag and islet inflammation.
    • Movie S3 (.avi format). Differential behavior of islet-specific CTLs and non–islet-specific CTLs at islets.
    • Movie S4 (.avi format). High amounts of CTLs not recognizing local Ag reduces the number of interactions with APCs.
    • Movie S5 (.avi format). High amounts of CTLs not recognizing local Ag reduces the number of interactions with APCs.

    Files in this Data Supplement:

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