PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods
Aim. Evidence-backed execution summary for PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods from PD-L1 regulates the development, maintenance, and function of induced regulatory T cells.
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PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells
reagent used in the protocol.
- Use
- To assess whether PD-L1-induced iT reg cells not only express Foxp3 but also function as suppressor T cells, we treated naive T cells with TGF-β plus control Ig or PD-L1-Ig beads and sorted Foxp3.GFP + iT reg cells after 3 d of culture. Sorted iT reg cells were then cultured in a standard suppressio...
PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells
reagent used in the protocol.
- Use
- PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells in vitro. (A) PD-L1 iT reg cell function was assessed by [ 3 H]thymidine incorporation of naive CD4 + CD25 - T eff cells after 3 d of co-culture at a 1:1 T reg/T eff cell ratio plus PD-L1 beads (5:1 bead/T eff cell ratio). Data represent...
PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios
reagent used in the protocol.
- Use
- Our studies thus far could not discriminate whether PD-L1 only controls iT reg cell development or also has a role in maintaining iT reg cell function. Recent studies indicate that continued Foxp3 expression is necessary for sustaining T reg cell function (; ). Therefore, we investigated whether PD-L1 influences th...
PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios
reagent used in the protocol.
- Use
- PD-L1 maintains Foxp3 expression by iT reg cells during suppression of effector cell function. (A) Schematic depiction of experiment. Naive CD4 + CD62L hi Foxp3.GFP - T cells were induced toward T reg cell differentiation for 3 d in the presence of TGF-β plus IL-2 and either control or PD-L1 beads. Foxp3....
PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios
reagent used in the protocol.
- Use
- We next tested whether the presence of PD-L1 could influence the efficiency of suppression by iT reg cells. Foxp3.GFP + iT reg cells were sorted and cultured with naive CD4 + CD25 - CD45.1 + T eff cells plus either PD-L1-Ig or control Ig beads at a variety of iT reg/T eff cell ratios, as graphically depi...
PD-L1 -/- PD-L2 -/- Rag -/- mice develop fatal immune-mediated pulmonary damage a...
reagent used in the protocol.
- Use
- To ascertain the critical role for PD-L1 in vivo, we transferred naive CD4 T cells to Rag -/- recipients treated with anti-PD-L1 blocking antibody ( Fig. S1 ) and monitored the mice for 3-4 wk. Mice were sacrificed to assess T reg cell development and immunopathology. A significant defect in...
PD-L1 antagonizes the Akt-mTOR signaling cascade during the induction of iT reg cells
reagent used in the protocol.
- Use
- Recent studies have shown notable differences in signaling pathways used by CD4 + T eff cells compared with T reg cells. In particular, Akt signaling is essential for naive T cell activation and proliferation but dispensible for T reg cell development and function (;;;;;; ). These findings led us to hypothesiz...
PD-L1 antagonizes the Akt-mTOR signaling cascade during the induction of iT reg cells
reagent used in the protocol.
- Use
- PD-L1 regulates T reg cell development by antagonizing the Akt-mTOR signaling cascade. (A, C, F, and G) Phospho-Akt, phospho-mTOR, PTEN, and phospho-S6 analysis at 18 h after culture with control Ig bead (hIgG = 60% of bead surface, with remaining surface coated with anti-CD3 and anti-CD28) or various titers o...
PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells
To assess whether PD-L1-induced iT reg cells not only express Foxp3 but also function as suppressor T cells, we treated naive T cells with TGF-β plus control Ig or PD-L1-Ig beads and sorted Foxp3.GFP + iT reg cells after 3 d of culture. Sorted iT reg cells were then cultured in a standard suppressio...
- Use
- To assess whether PD-L1-induced iT reg cells not only express Foxp3 but also function as suppressor T cells, we treated naive T cells with TGF-β plus control Ig or PD-L1-Ig beads and sorted Foxp3.GFP + iT reg cells after 3 d of culture. Sorted iT reg cells were then cultured in a standard suppressio...
PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells
PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells in vitro. (A) PD-L1 iT reg cell function was assessed by [ 3 H]thymidine incorporation of naive CD4 + CD25 - T eff cells after 3 d of co-culture at a 1:1 T reg/T eff cell ratio plus PD-L1 beads (5:1 bead/T eff cell ratio). Data represent...
- Use
- PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells in vitro. (A) PD-L1 iT reg cell function was assessed by [ 3 H]thymidine incorporation of naive CD4 + CD25 - T eff cells after 3 d of co-culture at a 1:1 T reg/T eff cell ratio plus PD-L1 beads (5:1 bead/T eff cell ratio). Data represent...
PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios
Our studies thus far could not discriminate whether PD-L1 only controls iT reg cell development or also has a role in maintaining iT reg cell function. Recent studies indicate that continued Foxp3 expression is necessary for sustaining T reg cell function (; ). Therefore, we investigated whether PD-L1 influences th...
- Use
- Our studies thus far could not discriminate whether PD-L1 only controls iT reg cell development or also has a role in maintaining iT reg cell function. Recent studies indicate that continued Foxp3 expression is necessary for sustaining T reg cell function (; ). Therefore, we investigated whether PD-L1 influences th...
PD-L1 deficiency leads to impaired T reg cell conversion in vivo
To investigate the role of PD-L1 in iT reg cell development and function in vivo, we compared iT reg cell conversion and function in Rag -/- mice because iT reg cells can spontaneously develop from naive T cells in a lymphopenic environment (;; ). We adoptively transferred naive CD4 + CD62L hi Foxp3.GF...
- Use
- To investigate the role of PD-L1 in iT reg cell development and function in vivo, we compared iT reg cell conversion and function in Rag -/- mice because iT reg cells can spontaneously develop from naive T cells in a lymphopenic environment (;; ). We adoptively transferred naive CD4 + CD62L hi Foxp3.GF...
PD-L1 antagonizes the Akt-mTOR signaling cascade during the induction of iT reg cells
Recent studies have shown notable differences in signaling pathways used by CD4 + T eff cells compared with T reg cells. In particular, Akt signaling is essential for naive T cell activation and proliferation but dispensible for T reg cell development and function (;;;;;; ). These findings led us to hypothesiz...
- Use
- Recent studies have shown notable differences in signaling pathways used by CD4 + T eff cells compared with T reg cells. In particular, Akt signaling is essential for naive T cell activation and proliferation but dispensible for T reg cell development and function (;;;;;; ). These findings led us to hypothesiz...
MATERIALS AND METHODS
6-8-wk-old WT C57BL/6 and CD45.1 (B6.SJL- Ptprc a Pepc b /BoyJ ) mice were purchased from The Jackson Laboratory. PD-L1 -/- PD-L2 -/- ( ) and PD-L1 -/- ( ) mice were generated in our laboratory. 2D2 TCR Tg mice Foxp3-IRES-GFP knockin mice (Foxp3.GFP; ) were generated in our...
- Use
- 6-8-wk-old WT C57BL/6 and CD45.1 (B6.SJL- Ptprc a Pepc b /BoyJ ) mice were purchased from The Jackson Laboratory. PD-L1 -/- PD-L2 -/- ( ) and PD-L1 -/- ( ) mice were generated in our laboratory. 2D2 TCR Tg mice Foxp3-IRES-GFP knockin mice (Foxp3.GFP; ) were generated in our...
Reagents.
The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), an...
- Use
- The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), an...
In vitro suppression assays.
For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cel...
- Use
- For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cel...
Statistical analysis.
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analysis of Foxp3 + T reg cell development, T eff cell proliferation, intracellular cytokine production, and phospho-flow cytometry was performed using Student's t tests in StatView (SAS Institute Inc). PD-L1-Ig titration, TGF-β titration, and percentage of weight loss were analyzed by A...
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Reagents.
The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), and IFN-γ PE (clone XMG1.2; BD). Anti-CD3 (clone 2C11) plus anti-CD28 (clone 37.51) were used for bead conjugation and were purchased from Bio X Cell. Cells were sorted on a FACSAria cell sorter (BD). Cell surface staining was performed at 4°C in FACS Buffer (1% FCS, PBS, 2 mM EDTA; Invitrogen). CFSE was purchased from Invitrogen.
In vitro iT reg cell development.
Anti-CD3 (clone 2C11; Bio X Cell) plus anti-CD28 (clone 37.51; Bio X Cell) were covalently attached to Dynabeads M450 glycidyl ether beads according to the manufacturer's directions (Invitrogen). We ensured equal loading of proteins during preparation by keeping constant the total amount of protein (antibodies and fusion proteins) at 5 µg per 10 7 beads as previously described (; ). In general, 10 7 beads were coated with 1 µg of anti-CD3 (20% of total protein), 1 µg of anti-CD28, and either 60% control human IgG1 (referred to control Ig beads; Bio X Cell) or 40% PD-L1-hIgG1 Fc fusion protein (referred to as PD-L1-Ig beads 40; R& D Systems) plus 20% control human IgG1 Fc. In some experiments, increasing amounts of PD-L1-Ig was used to coat the epoxy beads (20, 40, and 60% of total protein per 10 7 beads = 1, 2, and 3 µg of PD-L1-Ig per 10 7 beads...
In vitro iT reg cell development.
CD4 + CD62L + Foxp3 - naive T cells were cultured with beads at a fixed ratio of 1:5 (T cells/beads). In brief, 1-2 × 10 6 T cells were plated at 10 6 /ml in a 24-well flat-bottom tissue culture plate with beads in complete media consisting of RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), penicillin-streptomycin (100 U penicillin and 100 µg streptomycin; Invitrogen), 12 mM HEPES (Invitrogen), and 50 µM β-mercaptoethanol (Sigma-Aldrich) plus 2 ng/ml TGF-β (R&D Systems) and 20 U/ml rh-IL2 (R&D Systems) for 3 d at 37°C with 5% CO 2. In some experiments, sorted CD4 + CD25 - CD62L + CD44 low naive T cells were labeled with 1 µM CFSE before culture with beads and TGF-β for 3 d at 37°C with 5% CO 2. T cells were then stained for Foxp3 expression (eBioscience). To further evaluate the effect o...
In vitro suppression assays.
For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cell ratio) in 96-well flat-bottom plates (BD). 72 h later, CD4 + CD45.1 + T cells were gated and analyzed for CFSE dilution. Division index (defined as the mean number of divisions that a cell has undergone) was calculated using FlowJo Proliferation analysis software (Tree Star, Inc.). For CFSE dilution experiments interrogating the suppressive capacity of PD-L1-iT reg cells versus control iT reg cells, naive T cells were induced toward T reg cell development in vitro using PD-L1 or control beads in the presence of TGF-β and IL-2 for 3 d, as described in the previous sec...
Phospho-flow cytometry.
Naive CD4 + T cells were sorted from 2D2 Foxp3.GFP reporter mice and cultured with either PD-L1-Ig beads or control beads in the presence of 2 ng/ml TGF-β and 20 U/ml IL-2 for 18 h. Signaling molecules were assessed with antibodies against phospho-Akt Ser473 Alexa Fluor 647 (clone D9E), phospho-mTOR Ser24448 (clone 49F9), phospho-S6 Ser235/236 Alexa Fluor 647 (clone D57.2.2E), and PTEN Alexa Fluor 647 (clone 138G6; Cell Signaling Technology). Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). p-mTOR was detected with anti-rabbit Alexa Fluor 647 secondary (Invitrogen). Intracellular staining was performed as described in the manufacturer's protocol. In brief, T cells were collected and washed thoroughly with PBS in 96-well V-bottom plates. Cells were then fixed with 2% paraformaldehyde for 10 min at 37...
Measurement outputs
What raw and processed outputs should exist?
Our studies thus far could not discriminate whether PD-L1 only controls iT reg cell development or also has a role in maintaining iT reg cell function. Recent studies indicate t...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
PD-L1 maintains Foxp3 expression by iT reg cells during suppression of effector cell function. (A) Schematic depiction of experiment. Naive CD4 + CD62L hi Foxp3.GFP - T ce...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We next tested whether the presence of PD-L1 could influence the efficiency of suppression by iT reg cells. Foxp3.GFP + iT reg cells were sorted and cultured with naive CD4 + CD...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
PD-L1 enhances the efficiency of iT reg cell-mediated suppression of T eff cells. (A) Foxp3.GFP + iT reg cells were sorted and co-cultured with naive CD4 + CD25 - CD...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To assess whether PD-L1-induced iT reg cells not only express Foxp3 but also function as suppressor T cells, we treated naive T cells with TGF-β plus control Ig or PD-L1-Ig beads and sorted Foxp3.GFP + iT reg cells after 3 d of culture.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Our studies thus far could not discriminate whether PD-L1 only controls iT reg cell development or also has a role in maintaining iT reg cell function. Recent studies indicate t...; PD-L1 maintains Foxp3 expression by iT reg cells during suppression of effector cell function. (A) Schematic depiction of experiment. Naive CD4 + CD62L hi Foxp3.GFP - T ce...; We next tested whether the presence of PD-L1 could influence the efficiency of suppression by iT reg cells. Foxp3.GFP + iT reg cells were sorted and cultured with naive CD4 + CD...; PD-L1 enhances the efficiency of iT reg cell-mediated suppression of T eff cells. (A) Foxp3.GFP + iT reg cells were sorted and co-cultured with naive CD4 + CD25 - CD....
from paperStatistical comparison
To assess whether PD-L1-induced iT reg cells not only express Foxp3 but also function as suppressor T cells, we treated naive T cells with TGF-β plus control Ig or PD...; Strikingly, all PD-L1 -/- PD-L2 -/- Rag -/- recipients were moribund within 17 d after transfer of naive T cells ( n = 12) in marked contrast...; To ascertain the critical role for PD-L1 in vivo, we transferred naive CD4 T cells to Rag -/- recipients treated with anti-PD-L1 blocking antibody ( Fig. S1 )...; Statistical analysis of Foxp3 + T reg cell development, T eff cell proliferation, intracellular cytokine production, and phospho-flow cytometry was performed using Student...
from paperReporting output
Report representative outputs alongside summary comparisons for Our studies thus far could not discriminate whether PD-L1 only controls iT reg cell development or also has a role in maintaining iT reg cell function. Recent studies indicate t..., PD-L1 maintains Foxp3 expression by iT reg cells during suppression of effector cell function. (A) Schematic depiction of experiment. Naive CD4 + CD62L hi Foxp3.GFP - T ce..., We next tested whether the presence of PD-L1 could influence the efficiency of suppression by iT reg cells. Foxp3.GFP + iT reg cells were sorted and cultured with naive CD4 + CD..., PD-L1 enhances the efficiency of iT reg cell-mediated suppression of T eff cells. (A) Foxp3.GFP + iT reg cells were sorted and co-cultured with naive CD4 + CD25 - CD....
inferred from protocolStructured statistical methods
To assess whether PD-L1-induced iT reg cells not only express Foxp3 but also function as suppressor T cells, we treated naive T cells with TGF-β plus control Ig or PD...; Strikingly, all PD-L1 -/- PD-L2 -/- Rag -/- recipients were moribund within 17 d after transfer of naive T cells ( n = 12) in marked contrast...; To ascertain the critical role for PD-L1 in vivo, we transferred naive CD4 T cells to Rag -/- recipients treated with anti-PD-L1 blocking antibody ( Fig. S1 )...; Statistical analysis of Foxp3 + T reg cell development, T eff cell proliferation, intracellular cytokine production, and phospho-flow cytometry was performed using Student...
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Evidence quotes (5)
The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), and IFN-γ PE (clone XMG1.2; BD). Anti-CD3 (clone 2C11) plus anti-CD28 (clone 37.51) were used for bead conjugation and were purchased from Bio X Cell. Cells were sorted on a FACSAria cell sorter (BD). Cell surface staining was performed at 4°C in FACS Buffer (1% FCS, PBS, 2 mM EDTA; Invitrogen). CFSE was purchased from Invitrogen.
Anti-CD3 (clone 2C11; Bio X Cell) plus anti-CD28 (clone 37.51; Bio X Cell) were covalently attached to Dynabeads M450 glycidyl ether beads according to the manufacturer's directions (Invitrogen). We ensured equal loading of proteins during preparation by keeping constant the total amount of protein (antibodies and fusion proteins) at 5 µg per 10 7 beads as previously described (; ). In general, 10 7 beads were coated with 1 µg of anti-CD3 (20% of total protein), 1 µg of anti-CD28, and either 60% control human IgG1 (referred to control Ig beads; Bio X Cell) or 40% PD-L1-hIgG1 Fc fusion protein (referred to as PD-L1-Ig beads 40; R& D Systems) plus 20% control human IgG1 Fc. In some experiments, increasing amounts of PD-L1-Ig was used to coat the epoxy beads (20, 40, and 60% of total protein per 10 7 beads = 1, 2, and 3 µg of PD-L1-Ig per 10 7 beads). In these cases, the remaining surface of the beads were coated with control human IgG1. Covalent attachment of the proteins to the beads was performed in NaPO 4 buffer for 24 h at room temperature on a Nutating Mixer (Lab-Tech Incorporated). Beads were then washed three times in PBS over a magnet...
CD4 + CD62L + Foxp3 - naive T cells were cultured with beads at a fixed ratio of 1:5 (T cells/beads). In brief, 1-2 × 10 6 T cells were plated at 10 6 /ml in a 24-well flat-bottom tissue culture plate with beads in complete media consisting of RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), penicillin-streptomycin (100 U penicillin and 100 µg streptomycin; Invitrogen), 12 mM HEPES (Invitrogen), and 50 µM β-mercaptoethanol (Sigma-Aldrich) plus 2 ng/ml TGF-β (R&D Systems) and 20 U/ml rh-IL2 (R&D Systems) for 3 d at 37°C with 5% CO 2. In some experiments, sorted CD4 + CD25 - CD62L + CD44 low naive T cells were labeled with 1 µM CFSE before culture with beads and TGF-β for 3 d at 37°C with 5% CO 2. T cells were then stained for Foxp3 expression (eBioscience). To further evaluate the effect of IL-2 on PD-L1-mediated T reg cell development, some experiments were conducted in the absence of exogenous rh-IL2 (as noted in the legend). To analyze the effect of TGF-β on PD-L1-mediated T reg cell development, we performed some experiments using a range of TGF-β concentrat...
For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cell ratio) in 96-well flat-bottom plates (BD). 72 h later, CD4 + CD45.1 + T cells were gated and analyzed for CFSE dilution. Division index (defined as the mean number of divisions that a cell has undergone) was calculated using FlowJo Proliferation analysis software (Tree Star, Inc.). For CFSE dilution experiments interrogating the suppressive capacity of PD-L1-iT reg cells versus control iT reg cells, naive T cells were induced toward T reg cell development in vitro using PD-L1 or control beads in the presence of TGF-β and IL-2 for 3 d, as described in the previous section, Foxp3.GFP + T cells were sorted on a FACSAria cell sorter. Control and PD-L Foxp3.GFP + iT reg cells were then co-cultured with CFSE-labeled CD4 + CD25 - Thy1.1 + T eff cells and stimulated with control beads (containing anti-CD3, anti-CD28, and control Ig). 10 5 CD4 + CD25 - Thy1....
Naive CD4 + T cells were sorted from 2D2 Foxp3.GFP reporter mice and cultured with either PD-L1-Ig beads or control beads in the presence of 2 ng/ml TGF-β and 20 U/ml IL-2 for 18 h. Signaling molecules were assessed with antibodies against phospho-Akt Ser473 Alexa Fluor 647 (clone D9E), phospho-mTOR Ser24448 (clone 49F9), phospho-S6 Ser235/236 Alexa Fluor 647 (clone D57.2.2E), and PTEN Alexa Fluor 647 (clone 138G6; Cell Signaling Technology). Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). p-mTOR was detected with anti-rabbit Alexa Fluor 647 secondary (Invitrogen). Intracellular staining was performed as described in the manufacturer's protocol. In brief, T cells were collected and washed thoroughly with PBS in 96-well V-bottom plates. Cells were then fixed with 2% paraformaldehyde for 10 min at 37°C. After fixation, plates were prechilled on ice for 1 min before permeabilization by slowly adding ice-cold methanol to a final concentration of 90% methanol. Cells were then incubated on ice for 30 min for permeabilization before being washed with 1% FCS/PBS (incubation buffer). Cells were b...
Machine-readable layer
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"@type": "HowTo",
"name": "PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods",
"description": "Evidence-backed execution summary for PD-L1 regulates the development, maintenance, and function of induced regulatory T cells methods from PD-L1 regulates the development, maintenance, and function of induced regulatory T cells.",
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"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Reagents.",
"text": "The following anti-mouse antibodies were used in cell surface staining, intracellular cytokine staining, and epoxy bead conjugation: anti-CD16/CD32 (Fc Block), CD4 PerCP-Cy5.5 (clone RM4-5), CD62L PE (clone MEL-14), IL-2 APC (clone JES6-5H4; eBioscience), CD45.1 APC (clone A20), IL-17 PE (clone TC11-18H10), and IFN-γ PE (clone XMG1.2; BD). Anti-CD3 (clone 2C11) plus anti-CD28 (clone 37.51) were used for bead conjugation and were purchased from Bio X Cell. Cells were sorted on a FACSAria cell sorter (BD). Cell surface staining was performed at 4°C in FACS Buffer (1% FCS, PBS, 2 mM EDTA; Invitrogen). CFSE was purchased from Invitrogen."
},
{
"@type": "HowToStep",
"position": 2,
"name": "In vitro iT reg cell development.",
"text": "Anti-CD3 (clone 2C11; Bio X Cell) plus anti-CD28 (clone 37.51; Bio X Cell) were covalently attached to Dynabeads M450 glycidyl ether beads according to the manufacturer's directions (Invitrogen). We ensured equal loading of proteins during preparation by keeping constant the total amount of protein (antibodies and fusion proteins) at 5 µg per 10 7 beads as previously described (; ). In general, 10 7 beads were coated with 1 µg of anti-CD3 (20% of total protein), 1 µg of anti-CD28, and either 60% control human IgG1 (referred to control Ig beads; Bio X Cell) or 40% PD-L1-hIgG1 Fc fusion protein (referred to as PD-L1-Ig beads 40; R& D Systems) plus 20% control human IgG1 Fc. In some experiments, increasing amounts of PD-L1-Ig was used to coat the epoxy beads (20, 40, and 60% of total protein per 10 7 beads = 1, 2, and 3 µg of PD-L1-Ig per 10 7 beads..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "In vitro iT reg cell development.",
"text": "CD4 + CD62L + Foxp3 - naive T cells were cultured with beads at a fixed ratio of 1:5 (T cells/beads). In brief, 1-2 × 10 6 T cells were plated at 10 6 /ml in a 24-well flat-bottom tissue culture plate with beads in complete media consisting of RPMI-1640 with L-glutamine (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich), penicillin-streptomycin (100 U penicillin and 100 µg streptomycin; Invitrogen), 12 mM HEPES (Invitrogen), and 50 µM β-mercaptoethanol (Sigma-Aldrich) plus 2 ng/ml TGF-β (R&D Systems) and 20 U/ml rh-IL2 (R&D Systems) for 3 d at 37°C with 5% CO 2. In some experiments, sorted CD4 + CD25 - CD62L + CD44 low naive T cells were labeled with 1 µM CFSE before culture with beads and TGF-β for 3 d at 37°C with 5% CO 2. T cells were then stained for Foxp3 expression (eBioscience). To further evaluate the effect o..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "In vitro suppression assays.",
"text": "For CFSE dilution experiments, CD4 + CD25 - CD45.1 + naive T eff cells were labeled with 1 mM CFSE for 10 min in RPMI-1640 (serum free) and washed twice with 100% FBS and twice with complete media before culture. 10 5 T eff cells were cultured with 10 5 iT reg cells and PD-L1-Ig beads (5:1 bead/T eff cell ratio) in 96-well flat-bottom plates (BD). 72 h later, CD4 + CD45.1 + T cells were gated and analyzed for CFSE dilution. Division index (defined as the mean number of divisions that a cell has undergone) was calculated using FlowJo Proliferation analysis software (Tree Star, Inc.). For CFSE dilution experiments interrogating the suppressive capacity of PD-L1-iT reg cells versus control iT reg cells, naive T cells were induced toward T reg cell development in vitro using PD-L1 or control beads in the presence of TGF-β and IL-2 for 3 d, as described in the previous sec..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Phospho-flow cytometry.",
"text": "Naive CD4 + T cells were sorted from 2D2 Foxp3.GFP reporter mice and cultured with either PD-L1-Ig beads or control beads in the presence of 2 ng/ml TGF-β and 20 U/ml IL-2 for 18 h. Signaling molecules were assessed with antibodies against phospho-Akt Ser473 Alexa Fluor 647 (clone D9E), phospho-mTOR Ser24448 (clone 49F9), phospho-S6 Ser235/236 Alexa Fluor 647 (clone D57.2.2E), and PTEN Alexa Fluor 647 (clone 138G6; Cell Signaling Technology). Isotype control staining was performed using rabbit IgG isotype mAb Alexa Fluor 647 (DA1E; Cell Signaling Technology). p-mTOR was detected with anti-rabbit Alexa Fluor 647 secondary (Invitrogen). Intracellular staining was performed as described in the manufacturer's protocol. In brief, T cells were collected and washed thoroughly with PBS in 96-well V-bottom plates. Cells were then fixed with 2% paraformaldehyde for 10 min at 37..."
}
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"name": "PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells"
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"name": "PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells"
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"name": "PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios"
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"name": "PD-L1 antagonizes the Akt-mTOR signaling cascade during the induction of iT reg cells"
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"name": "PD-L1-induced CD4 + Foxp3 + T reg cells suppress CD4 + T eff cells"
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"name": "PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios"
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"name": "PD-L1 enhances and maintains Foxp3 expression on iT reg cell and augments suppression at low T reg/T eff cell ratios"
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"name": "PD-L1 -/- PD-L2 -/- Rag -/- mice develop fatal immune-mediated pulmonary damage a..."
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"name": "PD-L1 antagonizes the Akt-mTOR signaling cascade during the induction of iT reg cells"
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"name": "PD-L1 antagonizes the Akt-mTOR signaling cascade during the induction of iT reg cells"
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"headline": "PD-L1 regulates the development, maintenance, and function of induced regulatory T cells",
"datePublished": "2009",
"author": [
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"@type": "Person",
"name": "Loise M. Francisco"
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{
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"name": "Victor H. Salinas"
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"name": "Keturah E. Brown"
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"name": "Vijay K. Vanguri"
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"name": "Vijay K. Kuchroo"
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"name": "Arlene H. Sharpe"
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"identifier": "10.1084/jem.20090847"
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