Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation methods
Aim. Evidence-backed execution summary for Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation methods from Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.
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mouse
Subject model for the experiment.
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Cell Isolation and Sorting.
reagent used in the protocol.
- Use
- Single cell suspensions were prepared from spleens, washed twice, and filtered through a fine nitex membrane. The samples were then enriched for either CD4 + cells with anti-CD4 magnetic microbeads or CD25 + cells with PE-anti-CD25 Ab and anti-PE magnetic beads using the MidiMACS ® system (Miltenyi Biotec...
Antibodies and FACS ®.
reagent used in the protocol.
- Use
- The following reagents were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2), allophycocyanin (APC)-anti-TCRαβ (H57-597), FITC- and PE-anti-CD4 (RM4-5), biotinylated anti-CD25 (7D4), PE-anti-CD25 (PC61), FITC-anti-CD44 (IM7), FITC-anti-CD45RB (16A), F...
MLR and Polyclonal Stimulation Assays.
reagent used in the protocol.
- Use
- Cultures were set up in triplicates in 96-well round-bottom plates (Costar) in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium with 10% FCS, 10 mM Hepes, 1% nonessential amino acids, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL), and 5 × 10 ͨ...
MLR and Polyclonal Stimulation Assays.
reagent used in the protocol.
- Use
- The culture conditions for the suppression assays with polyclonal anti-CD3 stimulation differed as follows: 5 × 10 4 TCD irradiated splenocytes were incubated with 2.5 × 10 4 syngeneic FACS ® -sorted CD4 + CD25 - and/or 2.5 × 10 4 CD4 + CD25 + T cells in the presence of 0.5 µg/ml anti-...
Cell Isolation and Sorting.
Single cell suspensions were prepared from spleens, washed twice, and filtered through a fine nitex membrane. The samples were then enriched for either CD4 + cells with anti-CD4 magnetic microbeads or CD25 + cells with PE-anti-CD25 Ab and anti-PE magnetic beads using the MidiMACS ® system (Miltenyi Biotec...
- Use
- Single cell suspensions were prepared from spleens, washed twice, and filtered through a fine nitex membrane. The samples were then enriched for either CD4 + cells with anti-CD4 magnetic microbeads or CD25 + cells with PE-anti-CD25 Ab and anti-PE magnetic beads using the MidiMACS ® system (Miltenyi Biotec...
Antibodies and FACS ®.
The following reagents were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2), allophycocyanin (APC)-anti-TCRαβ (H57-597), FITC- and PE-anti-CD4 (RM4-5), biotinylated anti-CD25 (7D4), PE-anti-CD25 (PC61), FITC-anti-CD44 (IM7), FITC-anti-CD45RB (16A), F...
- Use
- The following reagents were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2), allophycocyanin (APC)-anti-TCRαβ (H57-597), FITC- and PE-anti-CD4 (RM4-5), biotinylated anti-CD25 (7D4), PE-anti-CD25 (PC61), FITC-anti-CD44 (IM7), FITC-anti-CD45RB (16A), F...
MLR and Polyclonal Stimulation Assays.
Cultures were set up in triplicates in 96-well round-bottom plates (Costar) in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium with 10% FCS, 10 mM Hepes, 1% nonessential amino acids, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL), and 5 × 10 ͨ...
- Use
- Cultures were set up in triplicates in 96-well round-bottom plates (Costar) in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium with 10% FCS, 10 mM Hepes, 1% nonessential amino acids, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL), and 5 × 10 ͨ...
MLR and Polyclonal Stimulation Assays.
The culture conditions for the suppression assays with polyclonal anti-CD3 stimulation differed as follows: 5 × 10 4 TCD irradiated splenocytes were incubated with 2.5 × 10 4 syngeneic FACS ® -sorted CD4 + CD25 - and/or 2.5 × 10 4 CD4 + CD25 + T cells in the presence of 0.5 µg/ml anti-...
- Use
- The culture conditions for the suppression assays with polyclonal anti-CD3 stimulation differed as follows: 5 × 10 4 TCD irradiated splenocytes were incubated with 2.5 × 10 4 syngeneic FACS ® -sorted CD4 + CD25 - and/or 2.5 × 10 4 CD4 + CD25 + T cells in the presence of 0.5 µg/ml anti-...
CD4 + CD25 + T reg Cells Are Highly Enriched within the BM T Cell Population.
We previously showed that BM NK T cells, another T cell subpopulation with regulatory potential, are potent suppressors of lethal GVHD ( ). Although NK T cells are rare among T cells in secondary lymphoid organs such as the spleen, they are highly enriched within BM T cells. Unseparated BM T cells are only very weak...
- Use
- We previously showed that BM NK T cells, another T cell subpopulation with regulatory potential, are potent suppressors of lethal GVHD ( ). Although NK T cells are rare among T cells in secondary lymphoid organs such as the spleen, they are highly enriched within BM T cells. Unseparated BM T cells are only very weak...
CD4 + CD25 + T reg Cells Are Highly Enriched within the BM T Cell Population.
To clarify whether CD4 + CD25 + T cells in BM are also functionally comparable to CD4 + CD25 + T reg cells from other lymphoid organs, we purified TCRαβ + NK1.1 - CD4 + CD25 + cells from the BM of C57BL/6 animals and examined their proliferative response to polyclonal activation via CD3 in the presen...
- Use
- To clarify whether CD4 + CD25 + T cells in BM are also functionally comparable to CD4 + CD25 + T reg cells from other lymphoid organs, we purified TCRαβ + NK1.1 - CD4 + CD25 + cells from the BM of C57BL/6 animals and examined their proliferative response to polyclonal activation via CD3 in the presen...
CD4 + CD25 + T reg Cells Are Highly Enriched within the BM T Cell Population.
Suppressive effect of NK1.1 - CD4 + CD25 + T cells from the BM of C57BL/6 mice. (A) Suppression of the proliferation of 2.5 × 10 4 C57BL/6 splenic CD4 + CD25 - T cells, stimulated with soluble anti-CD3-Ab in the presence of autologous APC by 2.5 × 10 4 C57BL/6 NK1.1 - CD4 + CD25 + T...
- Use
- Suppressive effect of NK1.1 - CD4 + CD25 + T cells from the BM of C57BL/6 mice. (A) Suppression of the proliferation of 2.5 × 10 4 C57BL/6 splenic CD4 + CD25 - T cells, stimulated with soluble anti-CD3-Ab in the presence of autologous APC by 2.5 × 10 4 C57BL/6 NK1.1 - CD4 + CD25 + T...
CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...
Functional comparison of CD4 + CD25 + T cells from C57BL/6 WT and C57BL/6 IL-10 -/- animals. (A) Dose-dependent suppression of the alloresponses of C57BL/6 WT CD4 + CD25 - T cells to BALB/c stimulator cells by C57BL/6 WT or IL-10 -/- CD4 + CD25 + T cells. Cultures were set up with 10 5...
- Use
- Functional comparison of CD4 + CD25 + T cells from C57BL/6 WT and C57BL/6 IL-10 -/- animals. (A) Dose-dependent suppression of the alloresponses of C57BL/6 WT CD4 + CD25 - T cells to BALB/c stimulator cells by C57BL/6 WT or IL-10 -/- CD4 + CD25 + T cells. Cultures were set up with 10 5...
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MLR and Polyclonal Stimulation Assays.
Cultures were set up in triplicates in 96-well round-bottom plates (Costar) in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium with 10% FCS, 10 mM Hepes, 1% nonessential amino acids, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL), and 5 × 10 -5 M 2-mercaptoethanol (Sigma-Aldrich). Fixed numbers of responder cells and irradiated allogeneic stimulator cells (10 5 cells, respectively) were mixed with variable numbers of CD4 + CD25 + T cells to obtain the ratios indicated in the text and figures. After lysis of red blood cells, splenocytes were T cell depleted with anti-Thy1.2 magnetic beads using the MidiMACS ® system and irradiated with 3,000 cGy before they were used as stimulator cells. Proliferation was assessed after 5 d by pulsing the cells with 1 µCi/well [ 3 H]thymidine (Amersham Biosciences) fo...
Splenic CD4 + CD25 + T reg Cells Suppress Lethal aGVHD Induced by CD4 + CD25 - T Cells after Allogeneic Transpl...
aGVHD, induced by the transplantation of C57BL/6-derived splenocytes into lethally irradiated BALB/c hosts, is initiated predominantly by alloreactive CD4 + donor T cells ( ). To see whether naive CD4 + CD25 + T cells could suppress aGVHD induced by CD4 + CD25 - T cells, we coinjected the two subpopulations at a 1:10 and 1:1 ratio with TCD BM into BALB/c hosts within 24 h after lethal TBI (800 cGy; A). All mice that received 4.5 × 10 5 CD4 + CD25 - T cells and TCD BM developed signs of aGVHD (diarrhea, weight loss, and hunched posture) shortly after transplantation, and all died within 29 d. Mice given only TCD BM cells appeared healthy and 100% of the animals survived for at least 100 d. At a ratio of 1:10, which is similar to the percentage of CD4 + CD25 + and CD4 + CD25 - T cells in the spleen of normal C57BL/6 mice ( A), no protective effect of the CD4 + CD2...
CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...
Recently, it has been shown that IL-10 plays an important role in the suppression of some experimental autoimmune diseases and the facilitation of tolerance to alloantigens in vivo by CD4 + CD25 + T reg cells (,, ). However, this is in contrast to results obtained in vitro, where attempts to block the suppression of CD25 - by CD25 + T cells with anti-IL-4 or anti-IL-10 antibodies have failed (, - ). To further clarify the role of IL-10 production by T reg cells in the suppression of alloresponses in vitro and in vivo, we first compared the capacity of splenic CD4 + CD25 + T reg cells from WT and IL-10 -/- mice to suppress the proliferative response of WT splenic CD4 + CD25 - T cells to alloantigen in vitro. As shown in A, the proliferation of CD4 + CD25 - T cells from WT C57BL/6 animals in response to irradiated TCD BALB/c splenocytes...
CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...
Functional comparison of CD4 + CD25 + T cells from C57BL/6 WT and C57BL/6 IL-10 -/- animals. (A) Dose-dependent suppression of the alloresponses of C57BL/6 WT CD4 + CD25 - T cells to BALB/c stimulator cells by C57BL/6 WT or IL-10 -/- CD4 + CD25 + T cells. Cultures were set up with 10 5 CD4 + CD25 - T cells, 10 5 BALB/c stimulator cells, and variable numbers of CD4 + CD25 + T reg cells to obtain the indicated ratios. The bars represent the means of triplicate values and the brackets indicate the SDs. ***, P < 0.001; *, P < 0.05 (Student's t test). One of two experiments with similar results is shown. (B) Protection of BALB/c hosts from lethal aGVHD by CD4 + CD25 + T cells from C57BL/6 WT and IL-10 -/- animals. BALB/c mice received 800 cGy TBI, 2 × 10 6 C57BL/6 TCD BM cells, and 4.5 × 10 5 C57BL/6 WT CD4 + CD25 - T ce...
CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...
We then explored the requirement for T reg cell-derived IL-10 in the suppression of lethal GVHD by coinjecting splenic CD4 + CD25 - T cells from C57BL/6 WT mice with splenic CD4 + CD25 + T cells from IL-10 -/- or WT animals into lethally irradiated BALB/c hosts. Mice receiving additional IL-10-deficient T reg cells had a longer median survival time than those receiving WT CD4 + CD25 - T cells alone (45 vs. 15 d, respectively; P < 0.0001), but 60% of the mice eventually died within 60 d after transplant. These latter mice never regained the baseline body weight for animals receiving WT T reg cells as noted in B, and continued to show clinical signs of GVHD, including diarrhea. In contrast, mice that received additional WT CD4 + CD25 + T cells were completely protected from lethality (100% survival for >100 d) and returned to a normal appearance after...
Measurement outputs
What raw and processed outputs should exist?
The following reagents were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2), allophycocyanin (APC)-anti-TCRαβ (H57-597), FITC- and PE&...
- 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
Differences in the survival of the groups of hosts given BM transplants were analyzed using the log-rank test. Differences in the proliferation of responder cells were analyzed...
- 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 previously showed that BM NK T cells, another T cell subpopulation with regulatory potential, are potent suppressors of lethal GVHD ( ). Although NK T cells are rare among 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
Phenotypic characterization of CD4 + CD25 + NK1.1 - T cells from C57BL/6 BM and spleen. (A) Proportion of CD4 + CD25 + NK1.1 - cells among TCRαβ + cells in...
- 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
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Differences in the survival of the groups of hosts given BM transplants were analyzed using the log-rank test.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The following reagents were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2), allophycocyanin (APC)-anti-TCRαβ (H57-597), FITC- and PE&...; Differences in the survival of the groups of hosts given BM transplants were analyzed using the log-rank test. Differences in the proliferation of responder cells were analyzed...; We previously showed that BM NK T cells, another T cell subpopulation with regulatory potential, are potent suppressors of lethal GVHD ( ). Although NK T cells are rare among T...; Phenotypic characterization of CD4 + CD25 + NK1.1 - T cells from C57BL/6 BM and spleen. (A) Proportion of CD4 + CD25 + NK1.1 - cells among TCRαβ + cells in....
from paperStatistical comparison
Differences in the survival of the groups of hosts given BM transplants were analyzed using the log-rank test. Differences in the proliferation of responder cells were analyzed...; aGVHD, induced by the transplantation of C57BL/6-derived splenocytes into lethally irradiated BALB/c hosts, is initiated predominantly by alloreactive CD4 + donor T cells ( ). T...
from paperReporting output
Report representative outputs alongside summary comparisons for The following reagents were used for flow cytometric analysis: unconjugated anti-CD16/32 (2.4G2), allophycocyanin (APC)-anti-TCRαβ (H57-597), FITC- and PE&..., Differences in the survival of the groups of hosts given BM transplants were analyzed using the log-rank test. Differences in the proliferation of responder cells were analyzed..., We previously showed that BM NK T cells, another T cell subpopulation with regulatory potential, are potent suppressors of lethal GVHD ( ). Although NK T cells are rare among T..., Phenotypic characterization of CD4 + CD25 + NK1.1 - T cells from C57BL/6 BM and spleen. (A) Proportion of CD4 + CD25 + NK1.1 - cells among TCRαβ + cells in....
inferred from protocolStructured statistical methods
Differences in the survival of the groups of hosts given BM transplants were analyzed using the log-rank test. Differences in the proliferation of responder cells were analyzed...; aGVHD, induced by the transplantation of C57BL/6-derived splenocytes into lethally irradiated BALB/c hosts, is initiated predominantly by alloreactive CD4 + donor T cells ( ). T...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Cultures were set up in triplicates in 96-well round-bottom plates (Costar) in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium with 10% FCS, 10 mM Hepes, 1% nonessential amino acids, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL), and 5 × 10 -5 M 2-mercaptoethanol (Sigma-Aldrich). Fixed numbers of responder cells and irradiated allogeneic stimulator cells (10 5 cells, respectively) were mixed with variable numbers of CD4 + CD25 + T cells to obtain the ratios indicated in the text and figures. After lysis of red blood cells, splenocytes were T cell depleted with anti-Thy1.2 magnetic beads using the MidiMACS ® system and irradiated with 3,000 cGy before they were used as stimulator cells. Proliferation was assessed after 5 d by pulsing the cells with 1 µCi/well [ 3 H]thymidine (Amersham Biosciences) for the last 16 h. Cells were harvested onto filter membranes using a Wallac harvester (PerkinElmer) and the amount of incorporated [ 3 H]thymidine was measured with a Wallac Betaplate counter (PerkinElmer).
aGVHD, induced by the transplantation of C57BL/6-derived splenocytes into lethally irradiated BALB/c hosts, is initiated predominantly by alloreactive CD4 + donor T cells ( ). To see whether naive CD4 + CD25 + T cells could suppress aGVHD induced by CD4 + CD25 - T cells, we coinjected the two subpopulations at a 1:10 and 1:1 ratio with TCD BM into BALB/c hosts within 24 h after lethal TBI (800 cGy; A). All mice that received 4.5 × 10 5 CD4 + CD25 - T cells and TCD BM developed signs of aGVHD (diarrhea, weight loss, and hunched posture) shortly after transplantation, and all died within 29 d. Mice given only TCD BM cells appeared healthy and 100% of the animals survived for at least 100 d. At a ratio of 1:10, which is similar to the percentage of CD4 + CD25 + and CD4 + CD25 - T cells in the spleen of normal C57BL/6 mice ( A), no protective effect of the CD4 + CD25 + T cells could be seen. At a ratio of 1:1 of CD4 + CD25 + and CD4 + CD25 - T cells, however, the recipients were clearly protected from lethal aGVHD and 93% survived for 100 d (P < 0.0001; log-rank test). Animals receiving 9 × 10 5 CD4 + CD25 - T cells instead of the mixture of 4...
Recently, it has been shown that IL-10 plays an important role in the suppression of some experimental autoimmune diseases and the facilitation of tolerance to alloantigens in vivo by CD4 + CD25 + T reg cells (,, ). However, this is in contrast to results obtained in vitro, where attempts to block the suppression of CD25 - by CD25 + T cells with anti-IL-4 or anti-IL-10 antibodies have failed (, - ). To further clarify the role of IL-10 production by T reg cells in the suppression of alloresponses in vitro and in vivo, we first compared the capacity of splenic CD4 + CD25 + T reg cells from WT and IL-10 -/- mice to suppress the proliferative response of WT splenic CD4 + CD25 - T cells to alloantigen in vitro. As shown in A, the proliferation of CD4 + CD25 - T cells from WT C57BL/6 animals in response to irradiated TCD BALB/c splenocytes was suppressed in a comparable dose-dependent way by both WT and IL-10-deficient T reg cells, resulting in a >50% reduction in proliferation at a 1:4 ratio of CD25 + and CD25 - T cells (P < 0.05), and a >90% reduction (P < 0.001) at a ratio of 1:1. These data clearly indicate that IL-10...
Functional comparison of CD4 + CD25 + T cells from C57BL/6 WT and C57BL/6 IL-10 -/- animals. (A) Dose-dependent suppression of the alloresponses of C57BL/6 WT CD4 + CD25 - T cells to BALB/c stimulator cells by C57BL/6 WT or IL-10 -/- CD4 + CD25 + T cells. Cultures were set up with 10 5 CD4 + CD25 - T cells, 10 5 BALB/c stimulator cells, and variable numbers of CD4 + CD25 + T reg cells to obtain the indicated ratios. The bars represent the means of triplicate values and the brackets indicate the SDs. ***, P < 0.001; *, P < 0.05 (Student's t test). One of two experiments with similar results is shown. (B) Protection of BALB/c hosts from lethal aGVHD by CD4 + CD25 + T cells from C57BL/6 WT and IL-10 -/- animals. BALB/c mice received 800 cGy TBI, 2 × 10 6 C57BL/6 TCD BM cells, and 4.5 × 10 5 C57BL/6 WT CD4 + CD25 - T cells with or without 4.5 × 10 5 CD4 + CD25 + T cells from either C57BL/6 WT- or IL-10-deficient mice. Combined data from two independent experiments with 10 animals per group are shown.
We then explored the requirement for T reg cell-derived IL-10 in the suppression of lethal GVHD by coinjecting splenic CD4 + CD25 - T cells from C57BL/6 WT mice with splenic CD4 + CD25 + T cells from IL-10 -/- or WT animals into lethally irradiated BALB/c hosts. Mice receiving additional IL-10-deficient T reg cells had a longer median survival time than those receiving WT CD4 + CD25 - T cells alone (45 vs. 15 d, respectively; P < 0.0001), but 60% of the mice eventually died within 60 d after transplant. These latter mice never regained the baseline body weight for animals receiving WT T reg cells as noted in B, and continued to show clinical signs of GVHD, including diarrhea. In contrast, mice that received additional WT CD4 + CD25 + T cells were completely protected from lethality (100% survival for >100 d) and returned to a normal appearance after mild and transient signs of aGVHD ( B). These results demonstrate that protection from lethal GVHD in vivo is partially dependent on IL-10 production by donor CD4 + CD25 + T reg cells.
Machine-readable layer
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{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation methods",
"description": "Evidence-backed execution summary for Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation methods from Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation.",
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"name": "MLR and Polyclonal Stimulation Assays.",
"text": "Cultures were set up in triplicates in 96-well round-bottom plates (Costar) in a total volume of 200 µl. Cells were cultured in RPMI 1640 medium with 10% FCS, 10 mM Hepes, 1% nonessential amino acids, 2 mM l -glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL), and 5 × 10 -5 M 2-mercaptoethanol (Sigma-Aldrich). Fixed numbers of responder cells and irradiated allogeneic stimulator cells (10 5 cells, respectively) were mixed with variable numbers of CD4 + CD25 + T cells to obtain the ratios indicated in the text and figures. After lysis of red blood cells, splenocytes were T cell depleted with anti-Thy1.2 magnetic beads using the MidiMACS ® system and irradiated with 3,000 cGy before they were used as stimulator cells. Proliferation was assessed after 5 d by pulsing the cells with 1 µCi/well [ 3 H]thymidine (Amersham Biosciences) fo..."
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"name": "Splenic CD4 + CD25 + T reg Cells Suppress Lethal aGVHD Induced by CD4 + CD25 - T Cells after Allogeneic Transpl...",
"text": "aGVHD, induced by the transplantation of C57BL/6-derived splenocytes into lethally irradiated BALB/c hosts, is initiated predominantly by alloreactive CD4 + donor T cells ( ). To see whether naive CD4 + CD25 + T cells could suppress aGVHD induced by CD4 + CD25 - T cells, we coinjected the two subpopulations at a 1:10 and 1:1 ratio with TCD BM into BALB/c hosts within 24 h after lethal TBI (800 cGy; A). All mice that received 4.5 × 10 5 CD4 + CD25 - T cells and TCD BM developed signs of aGVHD (diarrhea, weight loss, and hunched posture) shortly after transplantation, and all died within 29 d. Mice given only TCD BM cells appeared healthy and 100% of the animals survived for at least 100 d. At a ratio of 1:10, which is similar to the percentage of CD4 + CD25 + and CD4 + CD25 - T cells in the spleen of normal C57BL/6 mice ( A), no protective effect of the CD4 + CD2..."
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"name": "CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...",
"text": "Recently, it has been shown that IL-10 plays an important role in the suppression of some experimental autoimmune diseases and the facilitation of tolerance to alloantigens in vivo by CD4 + CD25 + T reg cells (,, ). However, this is in contrast to results obtained in vitro, where attempts to block the suppression of CD25 - by CD25 + T cells with anti-IL-4 or anti-IL-10 antibodies have failed (, - ). To further clarify the role of IL-10 production by T reg cells in the suppression of alloresponses in vitro and in vivo, we first compared the capacity of splenic CD4 + CD25 + T reg cells from WT and IL-10 -/- mice to suppress the proliferative response of WT splenic CD4 + CD25 - T cells to alloantigen in vitro. As shown in A, the proliferation of CD4 + CD25 - T cells from WT C57BL/6 animals in response to irradiated TCD BALB/c splenocytes..."
},
{
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"name": "CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...",
"text": "Functional comparison of CD4 + CD25 + T cells from C57BL/6 WT and C57BL/6 IL-10 -/- animals. (A) Dose-dependent suppression of the alloresponses of C57BL/6 WT CD4 + CD25 - T cells to BALB/c stimulator cells by C57BL/6 WT or IL-10 -/- CD4 + CD25 + T cells. Cultures were set up with 10 5 CD4 + CD25 - T cells, 10 5 BALB/c stimulator cells, and variable numbers of CD4 + CD25 + T reg cells to obtain the indicated ratios. The bars represent the means of triplicate values and the brackets indicate the SDs. ***, P < 0.001; *, P < 0.05 (Student's t test). One of two experiments with similar results is shown. (B) Protection of BALB/c hosts from lethal aGVHD by CD4 + CD25 + T cells from C57BL/6 WT and IL-10 -/- animals. BALB/c mice received 800 cGy TBI, 2 × 10 6 C57BL/6 TCD BM cells, and 4.5 × 10 5 C57BL/6 WT CD4 + CD25 - T ce..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "CD4 + CD25 + T reg Cells from IL-10 -/- Animals Suppress the Proliferation of WT CD4 + CD25 - T Cel...",
"text": "We then explored the requirement for T reg cell-derived IL-10 in the suppression of lethal GVHD by coinjecting splenic CD4 + CD25 - T cells from C57BL/6 WT mice with splenic CD4 + CD25 + T cells from IL-10 -/- or WT animals into lethally irradiated BALB/c hosts. Mice receiving additional IL-10-deficient T reg cells had a longer median survival time than those receiving WT CD4 + CD25 - T cells alone (45 vs. 15 d, respectively; P < 0.0001), but 60% of the mice eventually died within 60 d after transplant. These latter mice never regained the baseline body weight for animals receiving WT T reg cells as noted in B, and continued to show clinical signs of GVHD, including diarrhea. In contrast, mice that received additional WT CD4 + CD25 + T cells were completely protected from lethality (100% survival for >100 d) and returned to a normal appearance after..."
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"name": "MLR and Polyclonal Stimulation Assays."
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