Research ArticleIMMUNOTHERAPY

CTLA-4–mediated transendocytosis of costimulatory molecules primarily targets migratory dendritic cells

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Science Immunology  31 May 2019:
Vol. 4, Issue 35, eaaw0902
DOI: 10.1126/sciimmunol.aaw0902
  • Fig. 1 Constitutive cycling of CTLA-4 in Treg.

    CD4 T cells from BALB/c LNs were cultured in the presence or absence of anti-CD3/anti-CD28 beads at a 2:1 (T cell:bead) ratio for 6, 12, or 24 hours and analyzed by flow cytometry. (A) Representative fluorescence-activated cell sorting (FACS) plots showing CTLA-4 expression by Tregs (CD4+Foxp3+) and Tconv (CD4+Foxp3). CTLA-4 was stained on intact cells at 4°C (surface), at 37°C for 2 hours (cycling), or on fixed and permeabilized cells (total). (B) Collated data showing means + SD (n = 3 to 4); **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, two-way ANOVA. ns, not significant. Data are representative of at least four independent experiments. Raw data for 12 and 24 hours are shown in fig. S1.

  • Fig. 2 TE by Tregs and Tconv in vitro.

    (A and B) CD4 T cells from BALB/c LNs were cocultured with CD80-GFP–expressing CHO cells at a 1:1 ratio in the absence of stimulation or with anti-CD3 Ab (0.8 μg/ml) for 6 or 24 hours. (A) Representative FACS plots showing total CTLA-4 expression and GFP uptake by Tregs (CD4+Foxp3+) and Tconv (CD4+Foxp3). (B) Collated data (n = 4) showing fraction of GFP+ cells. (C and D) Tregs purified by magnetic-activated cell sorting were cultured overnight with CD80-GFP–expressing CHO cells at 1:1 ratio, with or without anti-CD3 Ab; 25 nM BafA was added for the final 4 hours of culture. Donor CHO cells were removed by magnetic separation, and T cells were imaged by confocal microscopy at 20× magnification. (C) Confocal images representative of at least three independent experiments. (D) Scoring of confocal images. Each point in unstimulated (n = 446) and stimulated (n = 323) conditions represents an individual cell from 11 to 12 separate images. Plots show mean signal intensity of CD25 and GFP and number of GFP fluorescence maxima (representing distinct GFP-filled punctae) per cell. Graphs show means + SD (CD25 fluorescence and GFP fluorescence) and means ± SD (GFP+ punctae); ***P ≤ 0.001, ****P ≤ 0.0001, two-tailed paired (B) or unpaired (C) Student’s t tests. Data are representative of three independent experiments.

  • Fig. 3 TE is CTLA-4 dependent and constitutively active in effector Tregs.

    (A and B) CD4 T cells isolated from LNs of mixed BM chimeric mice containing CTLA-4–sufficient (WT), and CTLA-4–deficient (KO) cells were cocultured with CD80-GFP–expressing CHO cells at a 1:1 ratio for 6 hours in the presence of anti-CD3 Ab (0.8 μg/ml). Lysosomal degradation was inhibited with 25 nM BafA where indicated. (A) Representative FACS plots showing acquisition of CD80-GFP by WT and CTLA-4−/− Foxp3+ Tregs. (B) Collated data from at least three independent experiments (n = 6 to 9). Graph shows means ± SD; **P ≤ 0.01, ****P ≤ 0.0001, paired two-tailed Student’s t test. (C) CD4 T cells from BALB/c LNs (n = 5) were cocultured with CD80-GFP–expressing CHO cells at a 1:1 ratio for 6 hours in the presence of different anti-CD3 Ab concentrations. TAPI-2 (100 μM) was added to inhibit shedding of CD62L. Graphs show the frequency of GFP+ cells within all Tregs (total Treg), CD45RB+CD62L+ Treg (resting Treg), or CD45RBCD62L (effector Treg) and are representative of two independent experiments.

  • Fig. 4 Preferential TE by Tregs in vivo.

    CD80-GFP–expressing mice were injected intravenously with 5 × 106 to 10 × 106 CD4 T cells from DO11 × RIP-mOVA mice and immunized with OVA/alum 24 hours later. Seven days after T cell transfer, mice were challenged with OVA peptide for 6 hours, in the presence of chloroquine to inhibit lysosomal degradation (600 μg ip) for the last 3 hours. (A) Acquisition of CD80-GFP by DO11 Tconv (CD4+Foxp3) and DO11 Tregs (CD4+Foxp3+) from spleens of immunized or unimmunized mice. Plots are representative of at least three independent experiments. (B) Splenocytes were enriched for CD4+ T cells, stained for CD4 and CD25, and imaged at 20× magnification. Images are representative of at least four independent experiments. (C) t-SNE dimensionality reduction analysis of CD3+CD4+ T cells in the immunized setting. GFP+ cells are highlighted by the black gate. Color axes show median expression of GFP, Foxp3, CD25, CTLA-4, DO11, and ICOS in each cell.

  • Fig. 5 Ligand capture by Tregs in response to tissue-expressed self-antigen.

    (A) DO11 Tconv (CD4+Foxp3) and Tregs (CD4+Foxp3+) from spleens, peripheral LN (pLN; axillary, brachial, inguinal, and cervical), pancreatic LNs (panLNs), and the pancreas of 12-week-old DO11 × RIP-mOVA mice (n = 3) were stained for intracellular CTLA-4 expression and analyzed by flow cytometry. Graphs show means + SD. (B) Representative FACS plots showing expression of ICOS and CTLA-4 in DO11 Tregs and Tconv in the pancreas. (C) Correlation of ICOS and CTLA-4 expression in Tregs and Tconv from lymphoid tissues and the pancreas of DO11 and DO11 × RIP-mOVA mice (n = 60 data points from six mice). Lines have been added to map linear relationships for visualization purposes. P value denotes comparison of the z-transformed r values. (D and E) CD80-GFP–expressing mice, or mock-transduced mice (GFP), with or without pancreatic expression of OVA were injected intraperitoneally with 5 × 106 to 10 × 106 CD4 T cells from DO11 × RIP-mOVA mice. Six days after T cell transfer, mice were injected with chloroquine (600 μg ip). (D) Representative FACS plots showing acquisition of CD80-GFP by DO11 Tregs and DO11 Tconv in the pancreas at day 7. (E) Collated data from three independent experiments showing means + SD. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, multiple t tests.

  • Fig. 6 CTLA-4–dependent modulation of cDC phenotype by polyclonal Tregs.

    CD80-GFP–expressing mice were injected intravenously with 5 × 106 to 10 × 106 BALB/c CD4 T cells. CTLA-4 was blocked by intraperitoneal injection of 500 μg of anti–CTLA-4 Ab every 2 to 3 days. Six days after T cell transfer, mice were injected with chloroquine (600 μg ip), and 24 hours later, splenocytes were analyzed by flow cytometry. (A) Representative FACS plots and (B) collated data showing acquisition of CD80-GFP by Tconv (CD4+Foxp3) and Tregs (CD4+Foxp3+) (n = 4 to 8). Data are representative of four independent experiments. (C) Frequency of the CD80+GFP+ population or (D) overall expression of CD80 within Lin-MHCII+CD11c+CD26+ cDCs in mice that received BALB/c CD4 T cells (+CD4) and controls (–CD4). Data show one representative experiment (n = 2) of two independent experiments. Graph shows means ± SD; **P ≤ 0.01, ****P ≤ 0.0001, unpaired two-tailed Student’s t test.

  • Fig. 7 Impact of CTLA-4 ablation on CD80 and CD86 expression in LN cDC subsets.

    (A and B) LNs (axillary, brachial, inguinal, and cervical) from 17- to 18-day-old CTLA-4−/− mice or CTLA-4+/− littermate controls were digested, and cells were stained for analysis by flow cytometry. (A) Representative FACS plots showing CD80 and CD86 expression on migratory and resident cDC subsets. (B) Collated data showing CD80 and CD86 expression on migratory and resident cDCs (n = 3). (C and D) BALB/c mice were treated with anti–CTLA-4 Ab or control IgG (Ctrl) and harvested after 1 or 4 days (d1 and d4, respectively) (1 or 2 doses of 500-μg anti–CTLA-4 Ab, respectively). LNs (axillary, brachial, inguinal, and cervical) were digested, and cells were stained for cDC markers and CD80 and CD86. (C) Representative FACS plots and (D) collated data (n = 3 to 4) are shown. Graphs show means + SD; **P ≤ 0.01, ****P ≤ 0.0001. Statistical significance was determined by two-way ANOVA. Data show one representative experiment of three independent experiments.

  • Fig. 8 Impact of T cell transfer on CD80 and CD86 expression in LN cDC subsets.

    Rag2−/− recipient mice were injected with 6 × 106 bulk CD4 T cells or 5.5 × 106 CD25-depleted CD4 T cells. Six days later, LNs (axillary, brachial, inguinal, and cervical) were digested, and cells were stained for analysis by flow cytometry. (A) Representative FACS plots showing CD80 and CD86 expression on migratory (Mig) and resident (Res) cDC subsets. (B) Collated data of CD80, CD86, and MHCII expression (n = 3 to 4). Graphs show means + SD; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, two-way ANOVA. Data are representative of at least four independent experiments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaaw0902/DC1

    Materials and Methods

    Fig. S1. Surface, cycling, and total CTLA-4 expression by Treg and Tconv.

    Fig. S2. Effects of CTLA-4 blockade and inhibition of lysosomal acidification on TE.

    Fig. S3. Internalization of CD80-GFP by CTLA-4–sufficient Treg.

    Fig. S4. TE by resting and effector Treg in response to different concentrations of anti-CD3 Ab.

    Fig. S5. CTLA-4 expression and cycling by resting and effector Treg.

    Fig. S6. CTLA-4–mediated down-regulation of CD80-GFP on BM-derived DCs.

    Fig. S7. GFP acquisition correlates with ICOS and CTLA-4 expression.

    Fig. S8. CTLA-4 expression is increased at sites of self-antigen recognition, and ICOS marks Treg with highest CTLA-4.

    Fig. S9. CTLA-4 targets expression of CD80 and CD86 on cDCs but not macrophages.

    Fig. S10. Gating strategy for identification of splenic cDC subsets.

    Fig. S11. Gating strategy for identification of LN resident and migratory cDC subsets.

    Fig. S12. Phenotype of Tconv and Tregs in CTLA-4–deficient mice.

    Fig. S13. CD80 and CD86 expression on splenic cDC subsets after CTLA-4 ablation.

    Fig. S14. Functional identification of migratory DCs by FITC skin painting.

    Fig. S15. Comparison of CD80 and CD86 expression in settings of CTLA-4 blockade and RAG deficiency.

    Fig. S16. CD80 and CD86 expression on splenic cDC subsets in Rag2-deficient mice with or without T cell transfer.

    Fig. S17. Impact of Treg expansion on CD80 and CD86 expression on cDC subsets.

    Table S1. Raw data file.

    Reference (63)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Surface, cycling, and total CTLA-4 expression by Treg and Tconv.
    • Fig. S2. Effects of CTLA-4 blockade and inhibition of lysosomal acidification on TE.
    • Fig. S3. Internalization of CD80-GFP by CTLA-4–sufficient Treg.
    • Fig. S4. TE by resting and effector Treg in response to different concentrations of anti-CD3 Ab.
    • Fig. S5. CTLA-4 expression and cycling by resting and effector Treg.
    • Fig. S6. CTLA-4–mediated down-regulation of CD80-GFP on BM-derived DCs.
    • Fig. S7. GFP acquisition correlates with ICOS and CTLA-4 expression.
    • Fig. S8. CTLA-4 expression is increased at sites of self-antigen recognition, and ICOS marks Treg with highest CTLA-4.
    • Fig. S9. CTLA-4 targets expression of CD80 and CD86 on cDCs but not macrophages.
    • Fig. S10. Gating strategy for identification of splenic cDC subsets.
    • Fig. S11. Gating strategy for identification of LN resident and migratory cDC subsets.
    • Fig. S12. Phenotype of Tconv and Tregs in CTLA-4–deficient mice.
    • Fig. S13. CD80 and CD86 expression on splenic cDC subsets after CTLA-4 ablation.
    • Fig. S14. Functional identification of migratory DCs by FITC skin painting.
    • Fig. S15. Comparison of CD80 and CD86 expression in settings of CTLA-4 blockade and RAG deficiency.
    • Fig. S16. CD80 and CD86 expression on splenic cDC subsets in Rag2-deficient mice with or without T cell transfer.
    • Fig. S17. Impact of Treg expansion on CD80 and CD86 expression on cDC subsets.
    • Reference (63)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

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