Increased T-bet+ cytotoxic effectors and type I interferon-mediated processes in chronic graft-versus-host disease of the oral mucosa

Abstract

Although chronic graft-versus-host disease (cGVHD) is a major long-term complication of allogeneic hematopoietic stem cell transplantation, little is known of its pathogenesis. We have systematically examined oral mucosa among cGVHD patients and determined that the clinical severity of oral cGVHD was correlated with apoptotic epithelial cells, often found adjacent to infiltrating effector-memory T cells expressing markers of cytotoxicity and type I cytokine polarization. Accumulation of T-bet(+) T-cell effectors was associated with both increased proliferation and the expression of the type I chemokine receptor CXCR3. Concurrently, in both infiltrating cells and keratinocytes, we observed increased expression of the CXCR3 ligand MIG (CXCL9) and interleukin-15 (IL-15), type I interferon (IFN)-inducible factors that support the migration, type I differentiation, and expansion of alloreactive effectors. In severely affected mucosa, we observed high levels of MxA, a protein specifically induced by type I IFN, and signal transducer and activator of transcription 1 (STAT1) phosphorylation, a critical step in the IFN-signaling pathway, along with increased numbers of plasmacytoid dendritic cells. These data challenge the current paradigm of cGVHD as a type II cytokine-driven disorder and support the model that oral cGVHD results from type I IFN-driven immigration, proliferation, and differentiation of T-bet(+) type I T effectors. The clinical trials are registered at http://www.clinicaltrials.gov as NCT00331968.

Figures

Figure 1

Clinical spectrum of oral GVHD, ranging from mild, predominantly lichenoid lesions to more severe, ulcerative lesions. (A) Lichenoid changes of the buccal mucosa. Note white striations (arrow). (B) Severe cGVHD of the buccal mucosa. Note pronounced erythema and the presence of ulcerations (arrow). (C,D) Mild and severe changes of the tongue.

Figure 2

Severe oral cGVHD is characterized by increased infiltration in the epithelial layer and apoptosis of keratinocytes. (A) Oral chronic GVHD is classically characterized by “dyskeratotic” epithelial cells and mononuclear cell satellitosis (small arrow) and lichenoid infiltrate (large arrow) under the basement membrane (dotted line), hematoxylin and eosin [H&E] × 40). Apoptotic cells (green) closely associated with infiltrating CD45 cells (red) are increased in the keratinocyte layer of the patient with severe oral GVHD (B) but not in the control patient lacking oral cGVHD symptoms (C). Apoptosis within epithelial layer is limited to keratinocytes. Cells within epithelial layer expressing active (cleaved) caspase-3 (D, red) also express cytokeratin (E, cyan). (F) Overlay. Both apoptotic cells (G) and CD45 cells (H) are significantly increased in the patients with severe disease (n = 8, n = 16, and n = 11 for control, mild, and severe groups, respectively; *P < .01; error bars represent SEM).

Figure 3

Infiltrating T cells express markers of cytotoxicity, type 1 cytokine polarization, and effector-memory phenotype. (A-D) Infiltrating T cells express CD45RO, a surface marker of the effector-memory T cells. Note predominance of the CD8 cells in the infiltrate. (E) CD68 myeloid cells are also overrepresented in oral cGVHD. CD8 cells express granzyme B (F) and Tia-1 (G) in the cytoplasm and are closely associated with the apoptotic cells (green, G). Note polarization of the cytotoxic granule (red) within the CD8 cell (cyan) in the direction of the apoptotic keratinocyte (green) shown by arrow. (B) Close-up of the same image with the arrows pointing to the granzyme B granules. (H,I) T-bet expression by the infiltrating T cells. (J-L) CD8 (n = 8, n = 16, and n = 12 for control, mild, and severe groups, respectively) and CD68 cells (n = 8, n = 15, and n = 10 for control, mild, and severe groups, respectively) are particularly prominent in severe disease (*P < .05; error bars represent SEM).

Figure 4

Migration and proliferation of infiltrating T cells in oral cGVHD. Infiltrating T cells (A) express the chemokine receptor CXCR3 (B). (C) Overlay of CD3 and CXCR3. (D) Increased production of the CXCR3 ligand MIG (CXCL9) by the keratinocytes (arrow) and infiltrating cells in a patient with severe oral GVHD. (E) Control patient without oral cGVHD symptoms with minimal MIG expression by the keratinocytes and endothelial cells (arrow). (F) Negative isotype staining control. (G) Enlarged image demonstrating MIG expression by CD68+ cells (large arrow), as well as CD68− cells (small arrow). Infiltrating T cells proliferate in the oral mucosa affected by cGVHD. (H) Severe oral cGVHD; close-up image showing nuclear expression of proliferation marker Ki-67 in a CD8 cell (large arrow). Basal keratinocytes (small arrow) proliferate constitutively. (I) Lack of proliferating T cells in a patient without clinical oral cGVHD. (J-L) Severe cGVHD (n = 12) is characterized by increased number and percentage of proliferating T cells compared with patients with mild (n = 16) or no (n = 8) oral cGVHD (*P < .05; error bars represent SEM).

Figure 5

Activation of type I IFN axis in oral cGVHD. (A) IL-15 expression in the keratinocytes and infiltrating cells in a patient with severe oral cGVHD. (B) Patient without oral cGVHD. (C) Negative isotype staining control. IL-15 mRNA (mean ± SEM) is increased in severe oral GVHD (n = 6) compared with mild cGVHD (n = 10) or controls (n = 5) and correlates with that of T-bet (D-E). Increased expression of MxA, IFN α/β–inducible protein, in oral cGVHD. (F) Patient with severe disease. (G) Control patient lacking oral cGVHD. (H) Negative isotype staining control. (I) MxA (MPI ± SEM) is markedly up-regulated in patients with severe oral cGVHD (n = 9) compared with those with mild disease (n = 7) or control patients (n = 6). MxA expression in the affected tissues correlates with the number of infiltrating T cells, assessed as CD3+ cells/HPF (J). STAT1 phosphorylation and nuclear translocation is observed in the patients with severe cGVHD (K), but not control patients (L). (M) Negative isotype staining control. (N) Plasmacytoid dendritic cells identified by expression of CD2ap in the patient with severe oral cGVHD and high type I IFN activity as measured by MxA expression. (O) Close-up of the image in panel N, showing coexpression of CD2ap and CD68 in a granular peripheral pattern. (P) No CD2ap-expressing cells and few CD68 cells are present in the mucosa of a patient without oral cGVHD. (Q) Negative control. AU indicates arbitrary units; MPI, mean pixel intensity.