Blocking TWEAK-Fn14 interaction inhibits hematopoietic stem cell transplantation-induced intestinal cell death and reduces GVHD

Abstract

Inhibition of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) system reduces intestinal cell death and disease development in several models of colitis. In view of the crucial role of TNF and intestinal cell death in graft-versus-host disease (GVHD) and the ability of TWEAK to enhance TNF-induced cell death, we tested here the therapeutic potential of Fn14 blockade on allogeneic hematopoietic cell transplantation (allo-HCT)-induced intestinal GVHD. An Fn14-specific blocking human immunoglobulin G1 antibody variant with compromised antibody-dependent cellular cytotoxicity (ADCC) activity strongly inhibited the severity of murine allo-HCT-induced GVHD. Treatment of the allo-HCT recipients with this monoclonal antibody reduced cell death of gastrointestinal cells but neither affected organ infiltration by donor T cells nor cytokine production. Fn14 blockade also inhibited intestinal cell death in mice challenged with TNF. This suggests that the protective effect of Fn14 blockade in allo-HCT is based on the protection of intestinal cells from TNF-induced apoptosis and not due to immune suppression. Importantly, Fn14 blockade showed no negative effect on graft-versus-leukemia/lymphoma (GVL) activity. Thus, ADCC-defective Fn14-blocking antibodies are not only possible novel GVL effect-sparing therapeutics for the treatment of GVHD but might also be useful for the treatment of other inflammatory bowel diseases where TNF-induced cell death is of relevance.

Figure 1

An ADCC-defective variant of the anti-Fn14 monoclonal antibody (mAb) 18D reduces acute GVHD and prolongs survival after allo-HCT. (A) Increased expression of Fn14 in intestinal epithelial cells of GVHD patients. Sections of normal mucosa (left) and mucosa with GVHD associated changes (middle and right) were stained with an anti-Fn14 antibody (ITEM-4) (shown in brown). Representative results are shown. Original magnification ×200. Fn14-positive immune cells are present in the lamina propria of both sample types, whereas Fn14-positive epithelial cells are only observed in GVHD samples. (B-D) Balb/c (H-2^d) mice were myeloablatively irradiated and transplanted with 5 × 10⁶ B6 (H-2^b) BM cells and 1 × 10⁶ enriched B6.L2G85.CD90.1 (H-2^b) T cells. Starting on day 1 posttransplantation, mice were treated daily with 100 µg of 18D1-dead, 18D1-enhanced, or an irrelevant hIgG1 control antibody. Shown are combined data from 3 independent experiments (18D1-dead: n = 20; 18D1-enhanced: n = 10; and hIgG1: n = 20). To control the efficacy of the myeloablative conditioning (irradiation only) and BM engraftment (BM control), mice were irradiated without transplantation or were irradiated and only transplanted with B6 (H-2^b) BM cells. (B) Survival of allo-HCT recipients. (C) Mice were weighed at the indicated times after allo-HCT. Weight measurements are shown in percent of initial weight. (D) Mice were assessed for clinical signs of GVHD at the indicated time points. Mean ± SEM. *P ≤ .05; **P ≤ .01 (hIgG1 vs 18D1-dead).

Figure 2

Treatment of allo-HCT recipients with the ADCC-defective anti-Fn14 mAb variant 18D1-dead does not affect donor T-cell target organ infiltration and cytokine production. Lethally irradiated Balb/c (H-2^d) mice were transplanted with 5 × 10⁶ B6 (H-2^b) BM cells alone or together with 1 × 10⁶ enriched B6.L2G85.CD90.1 (H-2^b) T cells. Mice of the latter group were treated daily with 100 µg of 18D1-dead or an irrelevant hIgG1 control antibody for 6 days. All mice (n = 5 per group) were then euthanized for ex vivo assessment of T-cell expansion, cytokine production, and cell death induction in the GI tract. (A) In vivo bioluminescence imaging of B6.L2G85.CD90.1 (H-2^b) cells in transplanted antibody-treated mice. Bioluminescence was imaged at indicated time points and light emission of donor T cells quantified. The upper panel shows the average light emission from ventral view and the lower panels show images from 1 representative mouse of each group. (B) Internal organs were analyzed ex vivo for donor T-cell–derived bioluminescence activity. Bioluminescence images of the organs of 1 representative mouse of each group are shown in the upper panel. The organ-derived averaged emissions are shown in the lower panel. (C) Large bowel biopsies of 18D1-dead and control hIgG1-treated GVHD mice and of untreated control mice were analyzed by qPCR for TNF expression. (D) Concentrations of various cytokines in serum were determined with the help of a cytometric bead array. Mean ± SEM. **P ≤ .01. cae, cecum; cLN, cervical lymph nodes; hea, heart; iLN, inguinal lymph nodes; ki, kidney; lb, large bowel; li, liver; lu, lung; mLN, mesenteric lymph nodes; sb, small bowel; sp, spleen; st, stomach; thy, thymus.

Figure 3

The ADCC-defective anti-Fn14 mAb variant 18D1-dead reduces allo-HCT–triggered and TNF-induced cell death of GI cells. (A) 3 µm intestinal tissue sections from mice shown in Figure 2 were analyzed by immunohistochemistry with a cleaved PARP1-specific antibody. Upper panel: graphic evaluation of apoptotic cells per field. Lower panel: representative photomicrographs. (B) Balb/c (H-2^d) were injected with 18D1-dead (200 µg in PBS), murine TNF (10 µg in PBS), a mixture of both, or with saline. After 6 hours, the mice were euthanized and small intestinal tissues were analyzed by immunohistochemistry for the presence of apoptotic cells with anticleaved caspase-3 (upper panel) and anticleaved lamin A-specific antibodies (lower panel). Mean ± SEM. *P ≤ .05; **P ≤ .01.

Figure 4

Treatment of allo-HCT recipients with the ADCC-defective anti-Fn14 mAb variant 18D1-dead delays the onset of acute GVHD while maintaining GVL effects. The effect of 18D1-dead on GVL activity was investigated in IM380 and A20 B-cell lymphoma models. Balb/c (H-2^d) mice were either injected with 10⁵ syngenic, luciferase-transgenic IM380 B-cell lymphoma cells iv 6 days before allo-HCT or with 10⁵ syngenic, luciferase-transgenic A20 cells along with allogeneic 5 × 10⁶ B6 (H-2^b) BM cells alone or together with 1 × 10⁶ enriched B6 (H-2^b) T cells. Starting on day 1 post–allo-HCT, mice were treated daily for a week with 100 µg of 18D1-dead or with 100 µg of an irrelevant hIgG1 mAb (n = 5 for each group). (A) In vivo bioluminescence imaging of luciferase⁺ lymphoma cells of a representative mouse of the indicated groups. Upper panels represent data at 8 days after transplantation; lower panels show images of a representative mouse of each group throughout the first 8 days of the experiment. (B) Survival analysis of allo-HCT recipients (right panel). Data from both GVL models are displayed, along with data from mice of the myeloablative conditioning control (irradiation only) and the BM engraftment control (BM control). Weight change after allo-HCT in percent of the initial weight (left panel). Statistics of hIgG1 vs 18D1-dead are indicated: mean ± SEM. *P ≤ .05; **P ≤ .01.