Donor-type CD4 + CD25 + Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation methods
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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...
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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.
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