Abstract
Regulatory T cells (Treg cells) can activate multiple suppressive mechanisms in vitro after activation via the T cell antigen receptor, resulting in antigen-independent suppression. However, it remains unclear whether similar pathways operate in vivo. Here we found that antigen-specific Treg cells activated by dendritic cells (DCs) pulsed with two antigens suppressed conventional naive T cells (Tnaive cells) specific for both cognate antigens and non-cognate antigens in vitro but suppressed only Tnaive cells specific for cognate antigen in vivo. Antigen-specific Treg cells formed strong interactions with DCs, resulting in selective inhibition of the binding of Tnaive cells to cognate antigen yet allowing bystander Tnaive cell access. Strong binding resulted in the removal of the complex of cognate peptide and major histocompatibility complex class II (pMHCII) from the DC surface, reducing the capacity of DCs to present antigen. The enhanced binding of Treg cells to DCs, coupled with their capacity to deplete pMHCII, represents a novel pathway for Treg cell–mediated suppression and may be a mechanism by which Treg cells maintain immune homeostasis.
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Data availability
The data that support the findings of this study are available from the corresponding author upon request.
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Acknowledgements
This research is funded by the Intramural Program, NIAID, NIH. We thank A. Athman and R. Kissinger (Visual Medical Arts, NIAID) for colorizing the electron microscopy images and various illustrations; A. Gangaplara, NIAID, for providing the SMARTA mice for the adoptive transfer experiments; A. Kumarpanda, NIAID, for providing the reagents for the in vivo Treg cell expansion; and M. Pena, NIAID, for technical assistance.
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B.A., Y.O., and E.M.S. conceived the project. E.M.S. secured the funding. B.A., M.A., Y.O., J.A.S., A.H.H., and O.K. performed the experiments. B.A., Y.O., M.A., J.K., and D.W.D. analyzed the data. D.D.G. provided technical help. B.A. and E.M.S. wrote the manuscript. M.A., Y.O., and R.M. edited the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Antigen-specific Treg cells perform bystander suppression in vitro.
DCs were loaded with 3 μM OVA323–339 (DCOVA), LCMV GP61–80 (DCSM) or both (DCOVA–SM). a-d) DCs (6 × 104) were cultured with CFSE labeled naive CD45.1+ OT-II or SMARTA T cells (3 × 105) in the presence of OT-II (a,b) or SMARTA (c,d) iTreg cells (1.5 × 104) for 3 days. e,f) DCs (6 × 104) were cultured with CFSE labeled naive CD45.1+ OT-II and SMARTA T cells (3 × 105) in the presence of OT-II iTreg cells (1.5 × 104) for 3 days. Graphs show the actual numbers of CD45.1+CFSElo proliferating cells. Bars indicate the means of n = 3 biological replicates (one per symbol); data are representative of two independent experiments. P values were calculated using two-sided Welch’s t-test.
Supplementary Figure 2 Antigen-specific tTreg cells perform antigen-specific suppression in vivo.
a-b) C57BL/6 DCs were loaded with 3 μM OVA323–339 and LCMV GP61–80 (DCOVA–GP). a) DCs (0.5 × 106), in vitro expanded tTreg cells (0.5 × 106), CFSE labeled OT-II (0.5 × 106) and SMARTA (0.5 × 106) Tnaive cells were transferred i.v. Flow cytometry plots demonstrate day 3 post-transfer proliferation and CD25 status of the Tnaive cells. Graphs show the number of CFSElo cells. b) DCs (2 × 104), freshly isolated tTreg cells (2 × 105), e450-labeled OT-II (1 × 105) and SMARTA (1 × 105) Tnaive were transferred i.v. Histograms demonstrate day 3 post-transfer proliferation status of the Tnaive cells. Graphs show the total number of cells. c) B10.A DCs (2 × 104) were loaded with 3 μM MCC88–103 and 3 μM HEL46–61, transferred i.v. with 5CC7 Tnaive cells (5 × 104) and freshly isolated 5CC7 or 3A9 tTreg cells (1 × 105). Lines mark the mean of n = 3 mice (one mouse per symbol). Data are representative of three independent experiments. P values were calculated using one-way ANOVA with Tukey’s correction.
Supplementary Figure 3 Antigen-specific iTreg cells have greater trogocytic capacity.
DCs were loaded with 3 μM MCC88–103 and co-cultured with 5CC7 or polyclonal Tnaive cells, Tactivated cells or iTreg cells for 18 h. a) Histograms demonstrate the surface levels for MHCII, B7 family members and ICAM-1 in the T cell gate upon co-culture with unpulsed DCs (Black) and antigen-pulsed DCs (Red). b) Histograms demonstrate the surface levels for MHCII, B7 family members and ICAM-1 in the T cell gate after 18 h culture in the absence (Gray tinted) or presence of 1 μg/ml plate-bound anti-CD3 and anti-CD28 (Red). c) Graphs show Δ MFI for the indicated surface molecules. Bars indicate the mean of n = 2 biological replicates (one per symbol), data are representative of three independent experiments. P values were calculated using one-way ANOVA with Tukey’s correction.
Supplementary Figure 4 Antigen-specific iTregs acquire higher levels of pMHCII over time.
DCs were loaded with 3 μM MCC88–103 and co-cultured 1:1 with 5CC7 Tnaive cells, 5CC7 Tactivated cells, 5CC7 iTreg cells or polyclonal iTreg cells for 3, 6 or 18 h. a) Histograms show the MCC88–103-I-Ek levels on live T cells. Gray tinted: T cells co-cultured with unpulsed DC, open histograms: T cells co-cultured with antigen pulsed DC. b) Graph shows the Δ MFI for MCC88–103-I-Ek in T cells. Bars indicate the mean of n = 2 biological replicates (one per symbol); data are representative of three independent experiments. P values were calculated using two-way ANOVA with Tukey’s post-test.
Supplementary Figure 5 CTLA-4 does not play role in pMHCII acquisition.
DCs were loaded with 3 μM MCC88–103 and co-cultured with WT or Ctla4–/– 5CC7 iTreg cells for 18 h. a) Histograms demonstrate the MCC88–103-I-Ek levels on the iTreg cell surface or in intracellular compartment, black: iTreg cells co-cultured with unpulsed DC (Ag-), red: iTreg cells co-cultured with antigen pulsed DC (Ag+). b) Histograms demonstrate the MCC88–103-I-Ek surface levels in the DC gate, gray tinted: unpulsed DC, black: antigen pulsed DC, red: antigen pulsed DC upon iTreg co-culture. Data are representative of three independent experiments, each performed in biological triplicates.
Supplementary Figure 6 Antigen-specific tTregs acquire specific pMHCII in vivo.
DCs from CD45.1+ B10.A animals were loaded with 3 μM MCC88–103 and/or 3 μM HEL46–61 or left unpulsed a-b) DCs (2 × 104) were co-cultured with freshly isolated e450 labeled 5CC7 (2 × 104) or 3A9 (2 × 104) tTreg cells for 18 h as in Figure 8. Graphs and histograms show the surface levels for captured MCC88–103-I-Ek and HEL46–61-I-Ak from double-pulsed DCs for 5CC7 (a) and 3A9 (b) tTreg cells. c-e) Double-pulsed DCs (2 × 105) and e450 labeled 3A9 tTreg cells (2 × 105) were adoptively transferred into WT B10.A mice i.v. Graphs show the tTreg cell surface (c), tTreg cell intracellular (d) and DC surface (e) levels of pMHCII 18 h post-transfer. Symbols show n = 2 biological replicates (a-b), n = 3 mice (c-e), n = 2 for no antigen group in e. Lines mark the mean, data are representative of two independent experiments. P values were calculated using one-way ANOVA with Tukey’s multiple comparison test (a,b,e) and two-sided Welch’s t-test (c,d).
Supplementary Figure 7 Antigen-specific iTreg cell treatment decreases DC function specifically.
DCs (4 × 106) were loaded with 3 μM MCC88–103 and 3 μM HEL46–61 (DCMCC–HEL) and cultured with e670 labeled 5CC7 iTreg cells (4 × 106), 3A9 iTreg cells (4 × 106) or alone for 18 h, then live e670–CD3ε– DCs were isolated by FACS sorting. Sorted DCs (2 × 104) were co-cultured with e450 labeled naive 5CC7 or 3A9 T cells (1 × 105) for 3 days. Histograms demonstrate the proliferation status of 5CC7 or 3A9 T cells. Data are representative of two independent experiments, each performed in biological triplicates.
Supplementary Figure 8 Working model for the antigen-specific Treg cell–mediated suppression via removal of cognate pMHCII complexes.
Antigen specific Treg cells engage into tight contacts with DC where they capture cognate pMHCII that also facilitates the acquisition of B7 family of ligands in close proximity while excluding non-cognate pMHCII. This results in the specific depletion of pMHCII on DC.
Supplementary information
Supplementary Video 1: Antigen-specific Treg cells engage into tight interactions with DCs
OVA323–339-pulsed splenic DCs from CD11c-YFP mice were cocultured with OT-II iTreg cells on Fibronectin-coated glass bottom dishes for 3 h. Cultures were stained with AF647 conjugated anti-CD4 at 37 °C for the last 30 min of the culture and visualized by confocal microscopy. 3D surfaces were generated for DCs (Blue) and iTreg cells (Red). Animation shows the interaction interface of DCs and iTreg cells as representative of five independent experiments
Supplementary Video 2a: Antigen-specific Treg cells display extensive filopodial movement during contact.
OVA323–339-pulsed splenic DCs from CD11c-YFP mice were cocultured with OT-II Tactivated cells (a) or OT-II iTreg cells (b) on Fibronectincoated glass bottom culture dishes for 3 h. Cultures were stained with AF647 conjugated anti-CD4 antibody at 37 °C for the last 30 min of the culture and imaged for real time DC-T cell interactions by confocal microscopy. Videos are representative of five independent experiments
Supplementary Video 2b: Antigen-specific Treg cells display extensive filopodial movement during contact.
OVA323–339-pulsed splenic DCs from CD11c-YFP mice were cocultured with OT-II Tactivated cells (a) or OT-II iTreg cells (b) on Fibronectincoated glass bottom culture dishes for 3 h. Cultures were stained with AF647 conjugated anti-CD4 antibody at 37 °C for the last 30 min of the culture and imaged for real time DC-T cell interactions by confocal microscopy. Videos are representative of five independent experiments
Supplementary Video 3a: Contact of antigen-specific Treg cells with DCs in vivo
OVA323–339 pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via footpad. The mice then received e450 labeled OT-II Tactivated cells or iTreg cells i.v. via retroorbital sinus. Popliteal lymph nodes of the recipient mice were imaged with intravital TP-LSM for starting at 18–20 h post-transfer. Videos demonstrate the in vivo interaction of antigen specific Tactivated cells (a) and iTreg cells (b) with DCs 18–20 h post-transfer. DCs: Green, T cells: Blue. Videos are representative of three independent experiments
Supplementary Video 3b: Contact of antigen-specific Treg cells with DCs in vivo
OVA323–339 pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via footpad. The mice then received e450 labeled OT-II Tactivated cells or iTreg cells i.v. via retroorbital sinus. Popliteal lymph nodes of the recipient mice were imaged with intravital TP-LSM for starting at 18–20 h post-transfer. Videos demonstrate the in vivo interaction of antigen specific Tactivated cells (a) and iTreg cells (b) with DCs 18–20 h post-transfer. DCs: Green, T cells: Blue. Videos are representative of three independent experiments
Supplementary Video 4a: Antigen-specific Treg cells abolish Tnaive cell clustering around DCs
OVA323–339 pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via the footpad. The mice then received naive OT-II-DsRed cells alone, with OT-II Tactivated cells or with OT-II iTreg cells i.v. via retroorbital sinus. Popliteal lymph nodes of the recipient mice were imaged with intravital TP-LSM starting at 18–20 h post-transfer. Videos show the movement and clustering of naive OT-II-DsRed (Red) cells around DCs (Green). a) OT-IIDsRed Tnaive cells + Unpulsed DCs), b) OT-II-DsRed Tnaive cells + OVA pulsed DCs, c) OT-II-DsRed Tnaive cells + OVA pulsed DCs + Tactivated cells, d) OT-II-DsRed Tnaive cells + OVA pulsed DCs + iTreg cells. Videos are representative of three independent experiments
Supplementary Video 4b: Antigen-specific Treg cells abolish Tnaive cell clustering around DCs
OVA323–339 pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via the footpad. The mice then received naive OT-II-DsRed cells alone, with OT-II Tactivated cells or with OT-II iTreg cells i.v. via retroorbital sinus. Popliteal lymph nodes of the recipient mice were imaged with intravital TP-LSM starting at 18–20 h post-transfer. Videos show the movement and clustering of naive OT-II-DsRed (Red) cells around DCs (Green). a) OT-IIDsRed Tnaive cells + Unpulsed DCs), b) OT-II-DsRed Tnaive cells + OVA pulsed DCs, c) OT-II-DsRed Tnaive cells + OVA pulsed DCs + Tactivated cells, d) OT-II-DsRed Tnaive cells + OVA pulsed DCs + iTreg cells. Videos are representative of three independent experiments
Supplementary Video 4c: Antigen-specific Treg cells abolish Tnaive cell clustering around DCs
OVA323–339 pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via the footpad. The mice then received naive OT-II-DsRed cells alone, with OT-II Tactivated cells or with OT-II iTreg cells i.v. via retroorbital sinus. Popliteal lymph nodes of the recipient mice were imaged with intravital TP-LSM starting at 18–20 h post-transfer. Videos show the movement and clustering of naive OT-II-DsRed (Red) cells around DCs (Green). a) OT-IIDsRed Tnaive cells + Unpulsed DCs), b) OT-II-DsRed Tnaive cells + OVA pulsed DCs, c) OT-II-DsRed Tnaive cells + OVA pulsed DCs + Tactivated cells, d) OT-II-DsRed Tnaive cells + OVA pulsed DCs + iTreg cells. Videos are representative of three independent experiments
Supplementary Video 4d: Antigen-specific Treg cells abolish Tnaive cell clustering around DCs
OVA323–339 pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via the footpad. The mice then received naive OT-II-DsRed cells alone, with OT-II Tactivated cells or with OT-II iTreg cells i.v. via retroorbital sinus. Popliteal lymph nodes of the recipient mice were imaged with intravital TP-LSM starting at 18–20 h post-transfer. Videos show the movement and clustering of naive OT-II-DsRed (Red) cells around DCs (Green). a) OT-IIDsRed Tnaive cells + Unpulsed DCs), b) OT-II-DsRed Tnaive cells + OVA pulsed DCs, c) OT-II-DsRed Tnaive cells + OVA pulsed DCs + Tactivated cells, d) OT-II-DsRed Tnaive cells + OVA pulsed DCs + iTreg cells. Videos are representative of three independent experiments
Supplementary Video 5: Antigen-specific exclusion of Tnaive cells from DCs
OVA323–339- and GP61–80-double-pulsed splenic DCs from CD11c-YFP mice were adoptively transferred into C57BL/6 mice via footpad. The mice then received naive OT-II-DsRed, naive e670-labeled SMARTA T cells and e450-labeled SMARTA iTreg cells (4 x 106) i.v. via retroorbital sinus. 18–20 h post-transfer, popliteal lymph nodes of the recipient mice were surgically removed, sectioned and recovered at 37 °C until the resumption of cell migration in the tissue. Sections were then imaged at 37 °C by confocal microscopy for 4 consecutive h. Video demonstrates the movement of SMARTA iTreg cells (Blue), OTII-DsRed Tnaive cells (Red) and SMARTA Tnaive cells (Magenta) around DCs (Green). White circle shows the DC interactions, white arrow points to an OT-II Tnaive cells, yellow arrows point to SMARTA Tnaive cells. Videos are representative of three independent experiments
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Akkaya, B., Oya, Y., Akkaya, M. et al. Regulatory T cells mediate specific suppression by depleting peptide–MHC class II from dendritic cells. Nat Immunol 20, 218–231 (2019). https://doi.org/10.1038/s41590-018-0280-2
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DOI: https://doi.org/10.1038/s41590-018-0280-2
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