Nuclear factor-kappaB inducing kinase is required for graft-versus-host disease

Abstract

Background: Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease.

Design and methods: We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control).

Results: We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells.

Conclusions: Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes.

Figure 1.

Expression of NIK in human graft-versus-host disease (GVHD). (A) Skin and (B) colon biopsies of a patient with acute GVHD. T lymphocytes positive for NIK expression were identified by immunohistochemical staining with an anti-NIK monoclonal antibody. Positive T lymphocytes are recognized by a dark-brown cytoplasmic and nucleic stain. (C) A biospy taken from a patient negative forGVHD showing no positive signal within the T lymphocytes, but only a slight background stain. The darkest spots correspond to melanophagocytes.

Figure 2.

GVHD across the major histocompatibility complex barrier did not develop with NIK-deficient T lymphocytes. Survival analysis of Balb/c mice transplanted with C57BL/6 or aly/aly cells (10⁷ marrow cells plus splenocytes containing 2×10⁶ CD8 cells). Results shown are from two independent experiments, with a total of 12 mice per group.

Figure 3.

T-cell expansion early during acute GVHD is impaired in aly/aly mice. (A) Purified T lymphocytes from spleens (Pre) were depleted of CD44-positive cells by immunomagnetic methods. The purity of depleted cells was assessed by flow cytometry (Post). (B) Donor T lymphocyte numbers recovered from the spleens of Balb/c mice transplanted with T cells (as explained in A) from C57BL/6 (solid bars) or aly/aly (open bars) mice. Note that Y labels are different at each time point analyzed. Representative of three experiments. (C) Total numbers of donor naïve (N), central memory (CM) and effector (E) T lymphocytes recovered from the spleens of Balb/c mice transplanted 120 h before with T cells (as explained in A) from aly/aly (open bars) or C57BL/6 (solid squares) mice. Each subpopulation is shown in more detail below. Results show the mean ± standard error, three mice per group. *P<0.05. **P<0.01.

Figure 4.

T-cell proliferation is maintained but apoptosis is enhanced early during acute GVHD in aly/aly mice. (A) Dilution of CFSE in donor T lymphocytes recovered from the spleens of Balb/c mice transplanted with purified T cells from aly/aly (top) or C57BL/6 (bottom) mice. Histograms represent CFSE intensity gated on donor cells. Representative of a single animal per group. (B) Proportions of annexin V-positive donor T lymphocytes recovered from the spleens of Balb/c mice transplanted with T cells from C57BL/6 (solid bars) or aly/aly (open bars) mice. Results shown are from two independent experiments, mean ± standard error, six mice per group per time point. *P<0.05.

Figure 5.

Th1 cytokine production is severely impaired in mice transplanted with aly/aly T lymphocytes. The levels of circulating IL-2, IL-12, IF-γ and TNF-α in Balb/c mice transplanted with C57BL/6 (black circles) or aly/aly (white circles) purified T lymphocytes. Three mice per time-point per group. Results show the mean ± standard error. *P<0.05.