Dichotomous regulation of GVHD through bidirectional functions of the BTLA-HVEM pathway - PubMed (original) (raw)

Dichotomous regulation of GVHD through bidirectional functions of the BTLA-HVEM pathway

Yukimi Sakoda et al. Blood. 2011.

Abstract

B and T lymphocyte attenuator (BTLA) is a co-inhibitory receptor that interacts with herpesvirus entry mediator (HVEM), and this interaction regulates pathogenesis in various immunologic diseases. In graft-versus-host disease (GVHD), BTLA unexpectedly mediates positive effects on donor T-cell survival, whereas immunologic mechanisms of this function have yet to be explored. In this study, we elucidated a role of BTLA in GVHD by applying the newly established agonistic anti-BTLA monoclonal antibody that stimulates BTLA signal without antagonizing BTLA-HVEM interaction. Our results revealed that provision of BTLA signal inhibited donor antihost T-cell responses and ameliorated GVHD with a successful engraftment of donor hematopoietic cells. These effects were dependent on BTLA signal into donor T cells but neither donor non-T cells nor recipient cells. On the other hand, expression of BTLA mutant lacking an intracellular signaling domain restored impaired survival of BTLA-deficient T cells, suggesting that BTLA also serves as a ligand that delivers HVEM prosurvival signal in donor T cells. Collectively, current study elucidated dichotomous functions of BTLA in GVHD to serve as a costimulatory ligand of HVEM and to transmit inhibitory signal as a receptor.

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Figures

Figure 1

Figure 1

Generation of agonistic, but not antagonistic, anti-BTLA mAb BYK-1. (A) T cells isolated from WT, _BTLA_-KO, or _HVEM_-KO mice were stimulated with indicated doses of anti-CD3 mAb in the presence of 20 μg/mL immobilized control rat IgG (□) or BYK-1 (■). Proliferative activity was assessed by 3H-thymidine incorporation during the last 18 hours of a 3-day culture. (B) BTLA-expressing CHO cells were cultured with HVEM-mouse Ig fusion protein in the presence of 10 μg control Ig, BYK-1, or BYK-2 (filled histogram). Background staining without HVEM-Ig is also shown (open histogram). Binding of HVEM-Ig fusion protein was detected by a staining with fluorescein isothiocyanate–conjugated antimouse Ig Ab. (C) CD4+ T cells isolated from B6, BALB/c, or DBA/2 mice were stimulated with 1 μg/mL antimouse CD3 mAb. After 48 hours, T cells were stained with BYK-1 (filled histogram) or control Ig (open histogram), and analyzed by flow cytometry. Data are representative of 3 independent experiments.

Figure 2

Figure 2

Inhibition of donor antihost alloresponses by BYK-1 treatment. (A-C) BDF1 recipient mice were injected intravenously with 5 × 107 donor B6 spleen cells. The recipient mice were treated intraperitoneally with 200 μg of BYK-1 (■) or control Ig (□) on days 0, 3, and 6. (A) On day 9, recipient spleen cells were harvested and assessed for CTL activity against P815 (H-2d) and EL4 (H-2b) cells by a standard 4-hours 51Cr releasing assay. (B) On day 9, recipient spleen cells were stained with anti-H-2Kd mAb, together with either anti-CD4 or anti-CD8 mAb, and analyzed by flow cytometry. Percentages of donor CD4+ or CD8+ T cells (upper left quadrant) in the recipient spleen are shown. The experiment was repeated more than 3 times, and representative data are shown. P < .05 (control Ig vs BYK-1). (C) On days 9 and 12, absolute numbers of donor CD4+ and CD8+ T cells in the recipient spleen were assessed. Each column represents average plus or minus SD of donor T-cell number after the treatment with control Ig (white bar) or BYK-1 (black bar). (D) BDF1 recipient mice were exposed to irradiation (9 Gy) and subsequently injected intravenously with 5 × 106 donor B6 spleen cells. The recipient mice were injected intraperitoneally with 200 μg BYK-1 (black bar) or control Ig (white bar) on days 0 and 3. On day 6, mesenteric lymph node cells harvested from the recipient mice were standardized for numbers of CD4+ T cells (5 × 104 per well) and stimulated in vitro with 5 μg/mL immobilized anti-CD3 mAb and 2 μg/mL anti-CD28 mAb. After 24 hours, the culture supernatants were harvested, and the levels of interferon-γ (left panel) and IL-4 (right panel) were measured by enzyme-linked immunosorbent assay. Each column represents average plus or minus SD. P < .05 (control Ig vs BYK-1). Data are representative of 3 independent experiments.

Figure 3

Figure 3

Therapeutic effects of BYK-1 in GVHD induced by MHC-matched, minor histocompatibility antigen-mismatched BMT. On day 0, B6 recipient mice were exposed to lethal irradiation (12 Gy) and transferred intravenously with 5 × 106 T cell-depleted BM cells together with 3 × 106 spleen plus lymph node cells isolated from C3H.SW donor mice. The recipient mice were treated intraperitoneally with 200 μg BYK-1 (▲) or control Ig (●) every 5 days from day 0 to day 25. As non-GVHD control, a group of mice were transferred with T cell–depleted BM cells alone (○). Survival of recipient mice (A), changes in body weight (B), and GVHD clinical scores (C) were monitored after BMT. (D) Pathologic features of recipient liver and skin were examined by hematoxylin and eosin staining (original magnification ×400 in the liver and ×100 in the skin). (E) Spleen cells of the recipient mice that had survived GVHD more than 100 days by BYK-1 treatment were assessed for the expression of Ly9.1 (donor cell marker) and CD3 or B220 by flow cytometry. Data are representative of 3 independently repeated experiments (n = 5 per group).

Figure 4

Figure 4

BTLA expressed on donor T cells as an essential target for inhibitory effects of BYK-1 in GVHD. (A) BDF1 mice were injected intravenously with 5 × 107 B6 WT or _BTLA_-KO mouse spleen cells on day 0 and treated intraperitoneally with 200 μg BYK-1 or control Ig on days 0, 3, and 6 in the following combinations: transfer of WT donor cells and control Ig treatment (□), transfer of WT donor cells and BYK-1 treatment (■), transfer of _BTLA_-KO donor cells and control Ig treatment (○), and transfer of _BTLA_-KO donor cells and BYK-1 treatment (●). On day 9, recipient spleen cells were harvested and analyzed for CTL activity against P815 (H-2d) and EL4 (H-2b) cells. (B) BDF1 mice were injected with mixture of T cells (2 × 107 cells) and non-T cells (4 × 107 cells) purified from either B6 WT or _BTLA_-KO mice and treated intraperitoneally with BYK-1 or control Ig on days 0, 3, and 6 in the following combinations: transfer of WT T cells plus _BTLA_-KO non-T cells followed by control Ig treatment (□), transfer of WT T cells plus _BTLA_-KO non-T cells followed by BYK-1 treatment (■), transfer of _BTLA_-KO T cells plus WT non-T cells followed by control Ig treatment (○), and transfer of _BTLA_-KO T cells plus WT non-T cells followed by BYK-1 treatment (●). On day 9, recipient spleen cells were harvested and analyzed for CTL activity against P815 (H-2d) and EL4 (H-2b). CTL level against P815 in BYK-1-treated WT T cells plus _BTLA_-KO non-T cells (■) was significantly lower than control Ig-treated _BTLA_-KO T cells plus WT non-T cells (○) at an effector/target (E/T) ratio of 100, 50, 25, and 12 (P < .05). Data are representative of 3 independently repeated experiments.

Figure 5

Figure 5

BTLA functions as a ligand to deliver prosurvival signal in GVHD. (A) Activated _BTLA_-KO B6 T cells expressing BTLAΔCY-GFP (black bar) or GFP alone (white bar) were transferred intravenously into sublethally (6 Gy)-irradiated BDF1 mice (7 × 105 cells/mouse). After 24 hours, spleen was harvested from the recipient mice and the numbers of GFP-positive donor T cells (total, CD4+, and CD8+) were assessed by flow cytometry. *P < .05. (B) Activated _BTLA_-KO B6 T cells expressing BTLAΔCY-GFP (black bar), wild-type BTLA-GFP (gray bar), and GFP alone (white bar) were incubated in vitro. After 4 and 7 days, the number of live GFP-positive cells was counted and percentage survival is shown as average plus or minus SD of triplicate wells. *P < .05. (C) T cells isolated from spleen of WT or _HVEM_-KO mice were labeled with CFSE and stimulated with 2 μg/mL anti-CD3 mAb in the presence of 10 μg/mL immobilized BTLA-Ig or control Ig. After 3 days, the intensity of CFSE of the culture cells was analyzed by flow cytometry. The numbers in panels indicate percentage of cells undergone more than one division.

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