B7-H3 expression in donor T cells and host cells negatively regulates acute graft-versus-host disease lethality by Rachelle G. Veenstra, Ryan Flynn, Katharina Kreymborg, Cameron McDonald-Hyman, Asim Saha, Patricia A. Taylor, Mark J. Osborn, Angela Panoskaltsis-Mortari, Annette Schmitt-Graeff, Elisabeth Lieberknecht, William J. Murphy, Jonathan S. Serody, David H. Munn, Gordon J. Freeman, James P. Allison, Tak W. Mak, Marcel van den Brink, Robert Zeiser, and Bruce R. Blazar Blood Volume 125(21):3335-3346 May 21, 2015 ©2015 by American Society of Hematology

B7-H3 expression is upregulated in target organs during acute GVHD B7-H3 expression is upregulated in target organs during acute GVHD. (A) B6-WT mice were lethally irradiated and infused with 107 BALB/c non-T-cell–depleted (NTCD) BM with or without 2 × 106 BALB/c-purified T cells (3 experiments were performed). B7-H3 expression is upregulated in target organs during acute GVHD. (A) B6-WT mice were lethally irradiated and infused with 107 BALB/c non-T-cell–depleted (NTCD) BM with or without 2 × 106 BALB/c-purified T cells (3 experiments were performed). (B) BALB/c mice were lethally irradiated and infused with 107 B6-WT BM only, B7-H3−/− BM only, or B7-H3−/− BM plus 1 × 106 purified B7-H3−/− T cells. Mice were euthanized on day 21 after transplantation, and the colon, liver, and lung were examined for B7-H3 mRNA by qPCR. Two experiments were performed with 5 mice per group per experiment; 1 representative experiment is depicted. (C) Representative colon sections of a patient without GVHD (left) and a patient with intestinal GVHD grade 4 (right) are shown. Red signal: detection of B7-H3+ cells. (D) Quantification of B7-H3 staining in intestinal sections from multiple patients is shown. *P < .05; **P < .01. ns, not significant. HPF, high power field. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology

B7-H3−/− recipients have accelerated GVHD lethality. B7-H3−/− recipients have accelerated GVHD lethality. (A) A mixed leukocyte reaction (MLR) was performed by coculturing BALB/c-purified T cells that were carboxyfluorescein diacetate succinimidyl ester (CFSE) –labeled with irradiated B6 or B7-H3−/− DC stimulators (10:1). Cells were analyzed by flow cytometry on day 9. Cells were gated on H2Kb-positive, viability dye–negative CD4+ or CD8+ events and were analyzed for dilution of CFSE (n = 5). One of 2 representative experiments is shown. (B) B6 or B7-H3−/− mice were lethally irradiated and infused with 107 BALB/c NTCD BM and 2 × 106 BALB/c-purified T cells. Survival plot of B6-WT (green solid circle) vs B7-H3−/− (red open circle) is shown (n = 16 per group; P < .0001). B6 or B7-H3−/− mice were lethally irradiated and infused with 107 BALB/c NTCD BM and 1 × 106 CD25-depleted BALB/c-purified T cells. Survival plot of WT (solid circle) vs B7-H3−/− (open circle) is shown (n = 16 per group pooled from 2 experiments with comparable results; P < .0001). (C) B6 or B7-H3−/− mice were lethally irradiated and infused with 107 BALB/c NTCD BM and 2 × 106 BALB/c-purified T cells. Mice were analyzed for clinical (left panel) and weight (right panel) scores (n = 8). One experiment was performed. (D) Mice were transplanted as in (C). Twenty-one days after transplant, colons were harvested, sectioned, and stained by hematoxylin and eosin (H&E). Sections were scored for pathology (n = 4). One of 2 representative experiments is shown. (E) B6 or B7-H3−/− mice were lethally irradiated and infused with 107 BALB/c NTCD BM and 1 × 106 BALB/c-purified T cells. Mice were sacrificed on day 7, and splenocytes were analyzed for percentage of α4β7, IL-2, and Ki-67 expression (n = 5). One experiment was performed. *P < .05; **P < .01; ***P < .001. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology

Increased gut injury in B7-H3−/− vs WT recipients. Increased gut injury in B7-H3−/− vs WT recipients. (A) B6-WT or B7-H3−/− mice were lethally irradiated and infused with 107 BALB/c NTCD BM and 3 × 106 BALB/c-purified CD25-depleted T cells. On day 21, 16 mg of FITC-dextran was administered orally to mice, and serum levels were measured 4 hours later (n = 4). One experiment was performed. (B) B6-WT or B7-H3−/− mice were lethally irradiated and infused with 107 BALB/c NTCD BM and 2 × 106 BALB/c-purified T cells. Mice were euthanized on day 21, and intraepithelial lymphocytes were analyzed for total cell numbers (n = 4; P < .05). Intraepithelial lymphocytes were analyzed for Ki-67 as well as effector cytokines IFN-γ, IL-17, and TNF-α (n = 4; P < .05). One experiment was performed. *P < .05; **P < .01. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology

B7-H3−/− donor T cells augment GVHD B7-H3−/− donor T cells augment GVHD. (A) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM and 1 × 106 B6-WT or B7-H3−/−–purified T cells. B7-H3−/− donor T cells augment GVHD. (A) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM and 1 × 106 B6-WT or B7-H3−/−–purified T cells. Survival plot of WT (solid circle) vs B7-H3−/− (open circle) is shown (n = 16 per group; P < .0001). Two experiments with comparable results were pooled. (B) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM and 1 × 106 B6 or B7-H3−/−-purified T cells. Mice were analyzed for weights and clinical scores, the latter of which were not scored beyond day 41 because they had reached mean values of >8 in both groups (n = 8 per group). One experiment was performed. (C) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM, 1 × 106 B6-purified T cells, and 0.5 × 106 B6-WT or B7-H3−/−-purified Tregs. Survival plot of B6-WT Tregs (solid triangle) vs B7-H3−/− Tregs (open triangle) is shown (n = 8-9 per group; P < .0001). One experiment was performed. (D) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM and 1 × 106 B6-WT or B7-H3−/− CD25-depleted–purified T cells. Survival plot of WT (solid circle) vs B7-H3−/− (open circle) is shown (n = 8 per group; P < .05). One experiment was performed. *P < .05; **P < .01. KO, knockout. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology

B7-H3−/− T cells have increased activation and proliferation and decreased apoptosis. B7-H3−/− T cells have increased activation and proliferation and decreased apoptosis. (A) MLR was performed by coculturing B6-WT or B7-H3−/−-purified T cells that were CFSE labeled with irradiated BALB/c DC stimulators (10:1). Cells were analyzed by flow cytometry on day 5. Cells were gated on H2Kb-positive, viability dye–negative CD4 or CD8 positive events and were analyzed for dilution of CFSE (n = 5). One of 2 representative experiments is shown. (B) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM and 1 × 106 B6-WT or B7-H3−/−-purified T cells. Mice were euthanized on day 7, and splenocytes were analyzed for total cell numbers (n = 4). One experiment was performed. (C) Splenocytes were analyzed for Ki-67, IFN-γ, IL-2, and annexin V α4β7 expression (n = 4). One experiment was performed. *P < .05; **P < .01; ***P < .001. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology

B7-H3−/− donor T cells have increased GVHD-induced gut injury. B7-H3−/− donor T cells have increased GVHD-induced gut injury. (A) BALB/c mice were lethally irradiated and infused with 107 B6-WT BM and 1 × 106 B6-WT or B7-H3−/−-purified T cells. On day 21, 16 mg of FITC-dextran was administered orally to mice, and serum levels were measured 4 hours later (n = 4). One experiment was performed. (B) BALB/c mice were lethally irradiated and infused with 107 B6 BM and 1 × 106 B6-WT or B7-H3−/−-purified T cells. Mice were euthanized on day 21 after transplant, and intraepithelial lymphocytes were analyzed for Ki-67 and annexin V expression (n = 4). One experiment was performed. (C) BALB/c mice were lethally irradiated and infused with 107 B6-WT BM and 1 × 106 B6-WT or B7-H3−/−- purified T cells. Mice were euthanized on day 21 after transplant, and intraepithelial lymphocytes and lamina propria lymphocytes were analyzed for CD4 or CD8 and IFN-γ, TNF-α, IL-2, or IL-17 coexpression (n = 4). One experiment was performed. *P < .05; **P < .01; ***P < .001. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology

B7-H3−/− splenocytes retain a GVL effect. B7-H3−/− splenocytes retain a GVL effect. (A) BALB/c mice were lethally irradiated and infused with 107 BALB/c T-cell-depleted BM. Mice were infused with phosphate-buffered saline (PBS) (no DLI) or either 30 × 106 B6-WT or B7-H3−/− splenocytes (Spl) (DLI; day 50). Three days later, the mice were infused with 106 A20luc tumor cells (n = 10 per group). One experiment was performed. No DLI vs B6-WT DLI, P = .0092; no DLI vs B7H3−/− DLI, P = .0014. (B) Mice were monitored for clinical scores that were not significant on day 30 or day 75 (n = 10 per group). One experiment was performed. No DLI vs WT or B7H3 −/− DLI, P ≤ .01. (C) BALB/c mice were lethally irradiated and infused with 107 B6 NTCD BM. Mice were then infused with 25 × 106 splenocytes (DLI; day 28) from B6-WT or B6-B7-H3−/− donors (n = 8 per group; 1 experiment was performed) or PBS (no DLI) as a control. Three days later, the mice were infused with 4 × 106 A20luc tumor cells (day 31). Survival after tumor infusion is shown (n = 7-8 mice per group; 1 experiment was performed). For PBS vs B6-WT DLI or B6-B7-H3−/− DLI, P < .01. Rachelle G. Veenstra et al. Blood 2015;125:3335-3346 ©2015 by American Society of Hematology