1. IL-21 blockade reduces graft-versus-host disease mortality by supporting inducible T regulatory cell generation
by Christoph Bucher, Lisa Koch, Christine Vogtenhuber, Emily Goren, Meghan Munger, Angela Panoskaltsis-Mortari, Pallavur Sivakumar, and Bruce R. Blazar
Blood
Volume 114(26):
December 17, 2009
©2009 by American Society of Hematology

2. Reduced GVHD lethality as a consequence of inhibiting IL-21/IL-21R signaling on donor T cells.
Reduced GVHD lethality as a consequence of inhibiting IL-21/IL-21R signaling on donor T cells. Survival and weight curves of lethally irradiated B10.BR recipients of T cell–depleted BM and Teffs from B6 mice. * indicates recipients of wt BM if not otherwise indicated. Recipients of 106 wt CD4+CD25− T cells treated with irrelevant IgG (▾) or anti–IL-21Ab (▿; P = .01; A-B). Recipients of CD4+CD25− T cells from wt (▾) and IL-21−/− (▿) donors (P < .001; C-D). Recipients of CD25− T cells from wt (▾) or IL-21−/− (▿) donors (P < .001; E-F). Recipients of IL-2γcR−/− BM (*) plus wt (▾) or IL-21−/− (▿) CD25− T cells (P < .001; C-D). Data from 2, 1, 4, and 1 experiments with 16, 8, 22, and 12 mice per group for panels A and B, C and D, E and F, and G and H, respectively.
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology

3. Reduced GVHD histopathology in recipients of IL-21−/− CD25− T cells.
Reduced GVHD histopathology in recipients of IL-21−/− CD25− T cells. (A) Lethally irradiated recipients were injected with BM only, BM + 2 × 106 wt CD25− T cells, or BM + IL-21−/− CD25− T cells, killed on day 14 after BMT, and hematoxylin and eosin–stained tissue sections were scored for GVHD: Colon (i,iii,v) and ileum (ii,iv,vi) from recipients of BM (i-ii), BM + wild-type T-effector cells (iii-iv) or BM + IL-21−/− cells (v-vi). Mean ± SEM of histologic grade of graft-versus-host disease in target organs in mice injected with BM only (□), BM + 2 × 106 wt CD25− T cells (■), or BM + IL-21−/− CD25− T cells (; wt vs IL-21−/−, P = .020 [colon] and P = .005 [small intestine], all others not significant [ns]; B).
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology

4. Comparative T-cell proliferation and apoptosis of IL-21−/− versus wt CD25− T cells during a GVHD response.
Comparative T-cell proliferation and apoptosis of IL-21−/− versus wt CD25− T cells during a GVHD response. Frequency of Ki-67–positive CD4+ and CD8+ spleen and lymph node T cells (A); mean fluorescence intensity (MFI) of annexin-V–allophycocyanin on CD4+ and CD8+ spleen and lymph node T cells (B). Total cell numbers in spleen and lymph nodes; (C) CD4/CD8 ratio in spleens and lymph nodes (D). All data at day 7 after BMT of lethally irradiated recipients injected with 107 T cell–depleted BM cells and 5 × 106 CD25− wt or IL-21−/− CD25− T cells. P = ns for all wt versus IL-21−/− comparisons. Data from 1 experiment with 3 mice per group.
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Effects of IL-21−/− CD25− T cells on cytokine and cytolytic Teff molecule production.
Effects of IL-21−/− CD25− T cells on cytokine and cytolytic Teff molecule production. Lethally irradiated recipients were injected with 2 × 106 wt of IL-21−/− CD25− T cells (triangles) or injected with 2 × 106 FoxP3− T cells and treated with irrelevant IgG or anti–IL-21 (diamonds). Colon lamina propria cells were isolated on day 14 and analyzed by FACS and the frequency of CD4+ cells expressing IFNγ (A), IL-4 (B), and IL-17 (C) is shown. The frequency of granzyme B (D) or TNFα (E) was determined on CD4+ (triangles) and CD8+ (diamonds) T cells. The frequency of CD4+ and CD8+ T cells coexpressing IFNγ/TNFα (F) or expressing FasL (G) is shown. P values are indicated. Data from 2 experiments (A,C) or 1 experiment (B,D-G) with 6 mice per group total.
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology

6. Effects of IL-21 on FoxP3+ T-cell generation.
Effects of IL-21 on FoxP3+ T-cell generation. Lethally irradiated B10.BR recipients were injected with 107 T cell–depleted bone marrow and (106 CD4+ (▴, ▵) or CD4+25− (▾, ▿) T cells from wt (▴,▾) or IL-21−/− (▵, ▿) mice and monitored for survival (A; P = .001 for wt vs IL-21−/− groups). Lethally irradiated recipients were injected with 2 × 106 wt of IL-21−/− CD25− T cells and lamina propria (LP) cells were isolated from the colon on day 14, stained with anti-CD4 and anti-FoxP3, and analyzed by FACS. The frequency of FoxP3-expressing CD4+ cells was determined (B; P = .002). Subgroups of mice described in panel A were killed on day 30 and sections from frozen tissue blocks were analyzed for expression of CD4 (green) and FoxP3 (red) by confocal microscopy (C). Lethally irradiated recipients were injected with 2 × 106 FoxP3− T cells from FoxP3-GFP knock-in mice. Subgroups were treated with irrelevant IgG or anti–IL-21 Ab. The frequency of FoxP3-expressing CD4+ spleen cells (D; P = .06) or CD4+LP T cells (E; P = .004) was determined. Data were obtained from 1 experiment each with 8 (A), 6 (B), 4 (C), and 6 (D-E) mice per group.
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Effect of donor IL-21 expression on the in vivo conversion of CD4+25− T cells into FoxP3+ cells.
Effect of donor IL-21 expression on the in vivo conversion of CD4+25− T cells into FoxP3+ cells. Weights (A,C) and survival (B,D) of lethally irradiated recipients injected with 107 T cell–depleted bone marrow and 0.5 × 106 flow-sorted CD45.1−CD4+CD25− T cells isolated from scurfy chimeric (A-B) or wt (C-D) donors. Subgroups were treated with irrelevant IgG (▾) or aIL-21 (▿). P values between treated and untreated groups for survival were .90 (B) and .12 (D). Data from 1 experiment with 8 mice per group.
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology

8. Loss of IL-21 signaling on donor T cells does not eliminate GVL activity.
Loss of IL-21 signaling on donor T cells does not eliminate GVL activity. Lethally irradiated Balb/c recipients were injected with T cell–depleted bone marrow and 105 A20 lymphoma cells on day 0. Subgroups received 2 × 106 CD25− T cells from wt or IL-21R−/− mice also on day 0. (A) Tumor growth was monitored by luciferase imaging on days 8, 20, and 32 after BMT. (B) Photon counts of BM-only recipients (*), recipients of BM + wt T-effector cells (▾), and BM + IL-21−/− cells (▿) on days 8, 20, and 32. (C) Survival of lethally irradiated Balb/c recipients injected with T cell–depleted bone marrow alone (*) or together with 2 × 106 CD25− T cells from wt B6 mice treated with irrelevant (irr) IgG (▾) or aIL-21 Ab (▵), or with 2 × 106 CD25− T cells from IL-21−/− (▿) or perforin−/− mice treated with irr IgG (♦) or aIL-21 Ab (◇; P < .001 for ▾ versus ▿, ▾ versus ♦, and ▾ versus ◇; P < .05 for ▾ versus ▿, ▿ versus ◇; P = ns for all others). (D) Survival of the same recipients as in panel C, but coinjected with 105 A20 lymphoma cells (P ≤ .001 for ▾ versus ▿; P < .05 for ▾ versus ▵, ♦ versus ◇, and ▾ versus ♦; P = ns for all others). Data from 2 experiments with a total of 8 (A-B) and 7 (C-D) mice per group.
Christoph Bucher et al. Blood 2009;114:
©2009 by American Society of Hematology