Critical role of host γδ T cells in experimental acute graft-versus-host disease by Yoshinobu Maeda, Pavan Reddy, Kathleen P. Lowler, Chen Liu, Dennis Keith Bishop, and James L. M. Ferrara Blood Volume 106(2):749-755 July 15, 2005 ©2005 by American Society of Hematology

Absence of γδ T cells in allogeneic BM transplant recipients reduces GVHD severity. Absence of γδ T cells in allogeneic BM transplant recipients reduces GVHD severity. B6 γδ-/- mice (γδ-/-: ▴, n = 24) or wild-type (wt: •, n = 38) mice received 1100 cGy TBI and underwent transplantation with 5 × 106 BM cells and 2 × 106 T cells from BALB/c or syngeneic B6 donors (syn: ○, n = 21) and evaluated for survival (A) and clinical GVHD score (B) as detailed in “Materials and methods.” Data from 4 similar experiments are combined. Error bars represent standard error. ▴ vs •, **P < .01 by Wilcoxon rank test. ▴ vs •, *P < .05 by Mann-Whitney U test. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology

In vivo depletion of γδ T cells from recipients reduces GVHD severity. In vivo depletion of γδ T cells from recipients reduces GVHD severity. Five days before BMT, mice were depleted of γδ T cells by intraperitoneal injection of anti-TCR γδ mAb. Three days later, iIELs were collected from mice treated with anti-TCR γδ mAb or control Ab and stained with PE-anti-γδ (A). γδ T-cell-depleted and sham-depleted mice received transplants from BALB/c (γδ T-cell-depleted: ▴, n = 13; sham-depleted: •, n = 13) or syngeneic B6 donors (γδ T-cell-depleted: ▵, n = 6; sham-depleted: ○, n = 6) as in Figure 1 and evaluated for survival (B) and clinical GVHD score (C). Data from 2 similar experiments are combined. Error bars represent standard error. ▴ vs •, **P < .01 by Wilcoxon rank test. ▴ vs •, *P < .05 by Mann-Whitney U test. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology

Absence of γδ T cells in the host reduces acute GVHD pathology Absence of γδ T cells in the host reduces acute GVHD pathology. γδ-/- (▥, n = 4), wt (▪, n = 4), and syn BM transplant recipients (□, n = 4) underwent transplantation as in Figure 1 and the gastrointestinal tract was analyzed on day 7 after BMT as described... Absence of γδ T cells in the host reduces acute GVHD pathology. γδ-/- (▥, n = 4), wt (▪, n = 4), and syn BM transplant recipients (□, n = 4) underwent transplantation as in Figure 1 and the gastrointestinal tract was analyzed on day 7 after BMT as described in “Materials and methods.” Damage to syn small bowel was minimal (A), whereas wt exhibited severe villous blunting, crypt destruction changes and atrophy, and increased lymphocytic infiltrates (B). γδ-/- small bowel showed significantly less damage (C). Original magnification, ×100. Images were visualized using an Olympus Bx 40 microscope (Olympus, Melville, NY) equipped with an 10 ×/0.65 aperture objective lens. Image acquisition was performed with a JVC digital camera GC-Qx 5HDU (JVC, Wayne, NJ). Coded slides were scored for pathologic damage (D) as described in “Materials and methods.” Serum was obtained on day 7 after BMT and analyzed for LPS (E) and TNF-α (F). γδ-/- vs wt, *P < .05 (D-F). Error bars represent standard error. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology

The host γδ T cells but not αβ T cells amplify the severity of GVHD The host γδ T cells but not αβ T cells amplify the severity of GVHD. (A,B) αβ-/- mice (▪, n = 11), γδ-/- mice (▴, n = 13), or wt mice (•, n = 10) received transplants from allogeneic BALB/c or syngeneic B6 donors (○, n = 10) as in Figure 1 and were evaluate... The host γδ T cells but not αβ T cells amplify the severity of GVHD. (A,B) αβ-/- mice (▪, n = 11), γδ-/- mice (▴, n = 13), or wt mice (•, n = 10) received transplants from allogeneic BALB/c or syngeneic B6 donors (○, n = 10) as in Figure 1 and were evaluated for survival (A) and clinical GVHD score (B). Data from 2 similar experiments are combined. ▴ vs ▪, **P < .01, ▪ vs •, P = .26 by Wilcoxon rank test. ▴ vs ▪, *P < .05, ▪ vs •, P = .43 by Mann-Whitney U test from weeks 1 to 5. Error bars represent standard error. (C,D) γδ+ splenocytes (, n = 12), αβ+ splenocytes (, n = 8) or media (▴ and solid line, n = 8) were injected into γδ-/- (60 × 106 cells/mouse). Wt B6 mice (• and solid line, n = 12) were injected with media alone. Twenty-four hours later, all mice received transplants from allogeneic BALB/c donors as in Figure 1 and were evaluated for survival (C) and clinical GVHD score (D). Data from 2 similar experiments are combined. vs , **P < .01 by Wilcoxon rank test. vs , weeks 3 to 5, *P < .001 by Mann-Whitney U test. Error bars represent standard error. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology

Absence of host γδ T cells reduces allogeneic donor T-cell responses. Absence of host γδ T cells reduces allogeneic donor T-cell responses. (A,B) γδ-/- (▥, n = 4), wt (▪, n = 4), and syn BM transplant recipients (□, n = 4) underwent transplantation as in Figure 1. Splenocytes were harvested on day 7 after BMT, and donor (H2Dd+) CD4+ T-cell (A) and CD8+ T-cell (B) phenotype were determined by FACS analysis as described in “Materials and methods.” Data represent the mean ± SE. wt vs γδ-/-, *P < .05. (C-E) Splenic DCs were isolated from γδ-/- (▴) and wt (•) mice 6 hours after 1100 cGy TBI and were cultured for 3 to 4 days with allogeneic responder BALB/c T cells. During the final 12 hours of a 96-hour culture, cells were pulsed with [3H] thymidine and assayed for proliferation (C). Supernatants were collected at 48 hours and 72 hours and assayed by ELISA for IL-2 (D) and IFN-γ (E), respectively, as described in “Materials and methods.” Each graph represents 1 of 4 similar experiments. wt vs γδ-/-, *P < .05. Error bars represent standard error. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology

γδ T cells activate DCs in vitro and in vivo. γδ T cells activate DCs in vitro and in vivo. Intestinal intraepithelial lymphocytes were sorted into γδ+ and γδ- populations. BM-derived DCs were then cultured with either γδ+ T cells (γδ+), γδ- T cells (γδ-), LPS, or medium. Allogeneic not T-cell proliferation was determined. DCs were isolated on a metrizamide gradient and the expression of CD40 was evaluated by FACS (B). The solid line denotes CD40 expression. The dashed line denotes the IgG negative control. These DCs were irradiated and then cultured with allogeneic BALB/c responder T cells that were assayed for proliferation (A). DCs plus γδ+ vs DCs plus γδ-, *P < .05. Responder BALB/c T cells were cultured for 3 to 4 days with irradiated B6 DCs that had been previously cultured for 36 hours with (C) wt γδ T cells with and without anti-IFN-γ mAb or with γδ T cells from IFN-γ-deficient (IFN-γ-/-) mice; (D) irradiated CD40-deficient (CD40-/-) DCs; (E) wt γδ T cells with and without anti-CD40L mAb (DCs plus γδ T) or separated by a 0.2 μ transwell membrane (DCs/γδ T transwell). During the final 12 hours of culture, cells were pulsed with [3H] thymidine and assayed for proliferation. DCs plus γδ T vs DCs/γδ T transwell, **P < .05. Each graph represents 1 of 3 to 4 similar experiments. Data represent the mean ± SE. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology

γδ T cells activate DCs and enhance allogeneic responses. γδ T cells activate DCs and enhance allogeneic responses. II-/- B6 mice were depleted of γδ T cells, irradiated with 1100 cGy TBI, and injected on day -1 with 5 × 106 B6 DCs that had been cocultured either with γδ T cells or with medium. Mice underwent transplantation with 5 × 106 T cells from bm12 donors on day 0. Splenocytes and serum were harvested on day 5 after BMT (n = 4 per group), and donor CD4+ T cells were phenotyped by FACS. Data represent the mean + SE. (A) Donor CD4+ T cells. ▥ vs ▪, *P < .05. (B) TNF-α. □ vs ▪, *P < .05. ND indicates not detected. Yoshinobu Maeda et al. Blood 2005;106:749-755 ©2005 by American Society of Hematology