Interleukin-10 spot-forming cells as a novel biomarker of chronic graft-versus-host disease

Abstract

Although there are National Institutes of Health consensus criteria for the global assessment of chronic graft-versus-host disease, no validated biomarkers have been established for this disease. Furthermore, whereas the role of T cells, B cells, and dendritic cells in chronic graft-versus-host disease has been established, the contribution of monocytes has not been clearly addressed. Using an enzyme-linked immunospot assay, we measured the spot-forming cells for interferon-γ, interleukin-4, interleukin-10, and interleukin-17 in unstimulated peripheral blood of patients following allogeneic hematopoietic stem cell transplantation. Other immunological examinations, including skin biopsy, were also done. Fifty-seven patients were enrolled. Interleukin-10 spot-forming cells were evaluable for therapeutic monitoring in 16 patients with chronic graft-versus-host disease. The number of interleukin-10 spot-forming cells in patients with active chronic graft-versus-host disease was significantly higher than the number in those with no or inactive chronic graft-versus-host disease. Interleukin-10 was predominantly produced by monocytes. CD29 expression on monocytes in patients with active chronic graft-versus-host disease was elevated. The level of plasma fibronectin, a ligand of CD29, correlated with the number of interleukin-10 spot-forming cells. Immunohistochemical analysis of the skin in active chronic graft-versus-host disease showed that infiltrating CD29(+) monocytes might produce interleukin-10. A novel biomarker, interleukin-10 spot-forming cells, shows promise as both a diagnostic and prognostic indicator for chronic graft-versus-host disease, and may allow for early intervention prior to the onset of the disease. Measurement of interleukin-10 spot-forming cells would be helpful in clinical trials and in patients' management.

Figure 1.

The clinical course of chronic GVHD and the cytokine SFC. The numbers of IFN-γ, IL-4, IL-10, and IL-17 SFC are shown, along with the clinical course of chronic GVHD in 16 patients. The sites and grading of chronic GVHD are depicted as gray bars according to the NIH chronic GVHD scoring system. Immunosuppressants, including calcineurin inhibitors (CNI), glucocorticoids (GC), and m y c o p h e n o l a t e mofetil (MMF), are shown as white bars. The shaded area shows the number of IL-10 SFC in PBMC obtained from healthy volunteers (n=35), which was used as the cut-off value for IL-10 SFC (mean+2SD). The number of cytokine SFC is shown per 50,000 PBMC.

Figure 2.

The value of IL-10 SFC derived from monocytes is a versatile biomarker for active chronic GVHD (cGVHD). (A) The numbers of IFN-γ, IL-4, IL-10, and IL-17 SFC are shown in patients with no cGVHD (white column; sample n=41), active cGVHD (black column; n=33), and inactive cGVHD (gray column; n=23). The number of cytokine SFC is shown per 50,000 PBMC. (B) PBMC obtained from seven patients with active cGVHD were separated with magnetic beads using negative selection into six subpopulations of cells before the ELISPOT assay. The data are expressed as mean±SD. The shaded area shows the cut-off value for IL-10 SFC (mean+2SD). **P<0.01.

Figure 3.

The levels of CD29 expression on monocytes in patients with active chronic GVHD (cGVHD). (A) PBMC gated with monocytoid light-scatter characteristics expressed CD14 and CD33 to more than 96% purity in all groups. The levels of CD29 expression on CD14⁺ monocyte-gated cells were compared in patients with no, active, and inactive cGVHD as representative data. (B) CD29⁺CD14⁺ cell populations were identified in patients with no cGVHD (n=9), active cGVHD (n=8), and inactive cGVHD (n=8). Data are expressed as mean±SD. *P<0.05.**P<0.01.

Figure 4.

The levels of plasma fibronectin in patients with active chronic GVHD (cGVHD). (A) Plasma levels of laminin, procollagen type I, and fibronectin were compared in patients with no (n=10), active (n=10), and inactive cGVHD (n=10). (B) The correlation of the number of IL-10 SFC with plasma laminin, procollagen type I, or fibronectin levels was analyzed in patients with active cGVHD. Data are expressed as mean±SD. *P<0.05.

Figure 5.

Immunohistochemical analysis of the skin of patients with chronic GVHD (cGVHD). Paraffin-embedded skin biopsy sections obtained from patients with no, active, and inactive cGVHD were stained with hematoxylin and eosin (H&E) (original magnifications ×100; A, F, and K) and immunostained for anti-human CD68 (×400; B, G, and L), IL-10 (×400; C, H, and M; ×1000 inset in H), CD29 (×400; D, I, and N), and fibronectin (×400;E, J, and O). Inset: the arrow indicates IL-10-producing mononuclear cells (MNC) (×1000). For the negative controls, either phosphate-buffered saline (×400; R) or normal goat serum (×400; S) was used as the primary antibody. Representative results from the indicated groups are shown (A-O). The mean numbers of infiltrating MNC per high power field (HPF) (×400) in patients with no (n=3), active (n=4), and inactive cGVHD (n=4) are shown (P). The mean numbers of CD68-positive, IL-10-positive, and CD29-positive MNC, per HPF (×400), are shown (Q). The data are expressed as mean±SD. *P<0.05. **P<0.01.