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. 2010 Jul 22;116(3):466-74.
doi: 10.1182/blood-2009-11-252825. Epub 2010 May 4.

Constitutively active Stat5b in CD4+ T cells inhibits graft-versus-host disease lethality associated with increased regulatory T-cell potency and decreased T effector cell responses

Christine Vogtenhuber  1 , Christoph Bucher, Steven L Highfill, Lisa K Koch, Emily Goren, Angela Panoskaltsis-Mortari, Patricia A Taylor, Michael A Farrar, Bruce R Blazar
Affiliations

Constitutively active Stat5b in CD4+ T cells inhibits graft-versus-host disease lethality associated with increased regulatory T-cell potency and decreased T effector cell responses

Christine Vogtenhuber et al. Blood. .

Abstract

Overexpression of a constitutively active form of Stat5b (Stat5b-CA) increases regulatory T cells (Tregs). We show that Stat5b-CA transgenic (TG) CD4(+) T cells had a markedly reduced graft-versus-host disease (GVHD) capacity versus wild-type (WT) T cells. Stat5b-CA TG versus WT CD4(+) T cells had a higher proportion of Tregs, which were superior in suppressing alloresponses mediated by CD4(+)CD25(-) effector T cells (Teffs). By day 5 after transplantation, Stat5b-CA TG Tregs had expanded approximately 3-fold more than WT Tregs. Purified Stat5b-CA TG Tregs added to WT CD4(+)CD25(-) Teffs were superior on a per-cell basis for inhibiting GVHD versus WT Tregs. Surprisingly, rigorously Treg-depleted Stat5b-CA TG versus WT CD4(+)CD25(-) Teffs caused less GVHD lethality associated with diminished Teff proinflammatory and increased Th2 anti-inflammatory cytokine responses. Reduced GVHD by Stat5b-CA TG versus WT Teffs could not be explained by conversion into Tregs in day 10 posttransplantation spleen or small intestine. In addition, Stat5b-CA TG Teffs retained a graft-versus-leukemia response. These results indicate a major role for Stat5 in Treg expansion and potency along with a lesser but significant role in Teff activation and suggest a strategy of pharmacologic Stat5b up-regulation as a means of decreasing GVHD while retaining a graft-versus-leukemia effect.

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Figures

Figure 1
Figure 1
Effect of Stat5b overexpression on CD4 T-cell function in a GVHD model. (A) CD25 and FoxP3 expression of pooled CD4+ T cells purified from LNs of WT (left) and Stat5b-CA TG (right) mice, used for the experiment shown in panel B. (B) Survival and weights of lethally irradiated B10.BR recipients injected with 107 BM (*) and 1 × 106 (▼, ▽) or 3 × 106 (♦, ◇) CD4+ T cells isolated from WT (▼, ♦) or Stat5b-CA TG (▽, ◇) mice. P = .003, ▼ vs ▽. P < .001, ♦ vs ◇. (C) Values are for one experiment; n = 8 mice/group. CD25 and FoxP3 expression of CD4+CD25+ T cells purified from pooled LNs of WT (left) and Stat5b-CA TG (right) mice, used for the experiment shown in panel D. (D) Survival and weights of lethally irradiated bm12 recipient injected with 107 BM (*) and 2 × 106 B6 CD4+CD25− T cells alone (▼) or along with 2 × 106 CD4+CD25+ Tregs isolated from WT (♦) or Stat5b-CA TG (◇) mice. P = .001, ♦ vs ◇. P < .001, ▼ vs ♦ and ◇. Pooled data are from 2 experiments; n = 16 mice/group total. (E) CD25 and FoxP3 expression of purified CD4+CD25+ T cells isolated from pooled LNs of WT (left) and Stat5b-CA TG (right) mice, used for experiment shown in panel F. (F) Survival and weights of lethally irradiated B10.BR recipients injected with 107 BM (*) and 1.5 × 106 B6 CD4+CD25− T cells alone (▼) or along with 1.125 × 106 CD4+CD25+ Tregs isolated from WT (♦) or Stat5b-CA TG (◇) mice. P = .003, ♦ vs ◇. P = .02, ▼ vs ♦. P < .001, ▼ vs ◇. Pooled data are from 2 experiments; n = 16 mice/group total. (G) Survival and weights of lethally irradiated B10.BR recipients injected with 107 BM (*) and 1.5 × 106 B6 CD4+CD25− T cells alone (▼) or along with 0.375 × 106 CD4+CD25+ Tregs isolated from Stat5b-CA TG (◇) mice. P = not significant, ▼ vs ◇. Pooled data are from 2 experiments; n = 16 mice/group total.
Figure 2
Figure 2
Effect of Stat5b-CA TG expression on Treg phenotype homeostasis and suppressor function. (A) Flow cytometric analysis of fresh WT and Stat5b-CA TG LN CD4+FoxP3+ T cells incubated with monoclonal antibody specific for the indicated Ag; representative data from 1 of 2 mice per group are shown. The percentage donor CD4+CD45.2+ expressing T cells in the spleen (B) and small intestine (C) isolated from lethally irradiated B10.BR recipients injected with 107 BM and 106 WT CD45.1+CD4+CD25− T cells together with WT or Stat5b-CA TG CD45.2+CD4+CD25+ Tregs on day 10 after transplantation. (D) Expression of IFN-γ and TNF-α in CD45.1+CD4+ T cells isolated from the small intestine of lethally irradiated B10.BR recipients injected with 107 BM and 106 WT CD45.1+CD4+CD25− T cells with or without WT or Stat5b-CA TG CD45.2+CD4+CD25+ Tregs on day 10 after transplantation. (B-D) P values are as indicated. Data are from 1 experiment with 4 to 6 mice/group.
Figure 3
Figure 3
Effect of Stat5b-CA TG expression on Teff phenotype and effector function. (A) CD4 and FoxP3 expression of freshly purified and pooled CD4+CD25− T cells isolated from LNs of 10 WT (left) and 6 Stat5b-CA TG (right) mice. (B) Survival and weights of lethally irradiated B10.BR recipients injected with 107 BM (*) and 2 × 106 CD4+CD25− T cells isolated from WT (▼) or Stat5b-CA TG mice (▽). P < .001, ▼ vs ▽. Pooled data are from 2 experiments; n = 16 mice/group total. (C) Flow cytometric analysis of freshly isolated WT and Stat5b-CA TG LN CD4+FoxP3− T cells incubated with monoclonal antibody specific for the indicated antigen; representative data from 1 of 2 mice per group are shown.
Figure 4
Figure 4
CFSE dilution and annexin V binding of CD4+CD25− T cells isolated from spleens of lethally irradiated B10.BR recipients injected with 107 BM and 5 × 106 CFSE-labeled CD4+CD25+ T cells from WT or Stat5b-CA TG mice. (A) Splenocytes were isolated on day 3 (left) and day 5 (right) after transplantation, and donor H-2Kb+CD4+ T cells were analyzed for CFSE dilution (illustrated in top and bottom panels) and annexin V (illustrated in top panels) expression. Representative flow cytometry data from one of 6 mice/group from 2 separate experiments are shown. Responder frequency (B) and proliferation index (C) were calculated. (D) Total cell numbers harvested from spleen on day 3 and day 5 from recipients described in panel A. (E) Survival of lethally irradiated B10.BR recipients of 107 BM (*) and 1 × 106 B6 CD4+CD25− T cells isolated from WT (▼) or Stat5b-CA TG (▽) mice or a mixture of both (●). P = not significant for ▼ vs ●. P < .01, ▼ and ● vs ▽. Pooled data are from 2 experiments; n = 16 mice/group total.
Figure 5
Figure 5
Stat5b-CA TG expression supports Treg proliferation after transplantation into allogeneic hosts. (A) CD4 and FoxP3 expression of purified CD4+CD25− T cells isolated from lymph nodes of WT (left) and Stat5b-CA TG (right) mice. Pooled data are from the experiment shown in panel B; n = 10/group. (B) Lethally irradiated B10.BR recipients were injected with 107 BM cells and 5 × 106 CFSE-labeled CD4+CD25− T cells from WT or Stat5b-CA TG mice. (B-C) Splenocytes were isolated on day 5 after transplantation, and the percentage and absolute number of CD4+H-2Kb+FoxP3+ WT and Stat5b-CA TG T cells were determined. P values are as indicated. (D) The CFSE dilution profile was determined. (E) The percentage host (H-2Kb−CD4+FoxP3+) and percentage donor (H-2Kb+CD4+FoxP3+) T cells were analyzed. (B-E) Representative data from one mouse or pooled data from 2 independent experiments of 6 or 7 mice/group total are shown.
Figure 6
Figure 6
Stat5b-CA TG expression impairs intrinsic Teff function but does not induce a Treg phenotype. (A) Survival and weights of lethally irradiated B10.BR recipients injected with 107 BM (*) and 1 × 106 sorted CD4+CD25−GFP− T cells from FoxP3-GFP-KI (WT; ▼) or FoxP3-GFP-KI × Stat5b-CA TG mice (TG; ▽) mice. P = .02, ▼ vs ▽. Pooled data are from 2 experiments; n = 16 mice/group total. (B) Histologic grade of GVHD in organs indicated on day 10 after transplantation in FoxP3-GFP-KI (WT; black) and FoxP3-GFP-KI × Stat5b-CA TG (Stat5b-CA TG; gray) recipients. **P < .01. *P < .05. Data are from 1 experiment with 4 and 7 or 8 mice/group. Splenocytes (C) and lamina propria cells (D) of recipients described in panel A were isolated on day 10, and the percentage FoxP3+(GFP+) cells was determined (panel C, 1 experiment with 4 and 5; and panel D, 4 and 8 mice per group, respectively). P values are as indicated. (E) The percentage TNF-α, IL-2, IFN-γ, IL-10, IL-17, or IL-4 expressing lamina propria CD4+ T cells isolated from the small intestine on day 10 after transplantation. P values are as indicated. Data are from 1 experiment with 4 and 7 or 8 mice/group.
Figure 7
Figure 7
Lethally irradiated B10.BR recipients were injected with 107 BM (*) and 1 × 106 CD4+CD25− T cells from WT (▼) or Stat5b-CA TG (●) mice. Mice were not challenged with A20 lymphoma cells and were monitored exclusively for GVHD lethality (A) and weights (B). Other cohorts of mice treated as in panel A also were given 105 A20 lymphoma cells on day 0 of BMT and were then monitored for survival (C) and weights (D). P values are as indicated. Data are from 1 experiment with 10 mice per group. Separate cohorts of mice were analyzed for bioluminescent imaging on days 7, 14, and 21. Data shown are the bioluminescence emissions (E) and the quantification of bioluminescence based on photons/sec/cm2 emission (y-axis) and days after BMT (x-axis; F). The SD of the mean is also shown.
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