Graft-versus-host disease: role of inflammation in the development of chromosomal abnormalities of keratinocytes

Abstract

Graft-versus-host disease (GVHD) is a major risk factor for secondary malignancy after hematopoietic stem cell transplantation. Squamous cell carcinoma (SCC) of the skin and mucous membranes are especially frequent in this setting where aneuploidy and tetraploidy are associated with aggressive disease. The current study is directed at the mechanism of neoplasia in this setting. Unmanipulated keratinocytes from areas of oral GVHD in 9 patients showed tetraploidy in 10% to 46% of cells when examined by florescein in situ hybridization (FISH). Keratinocytes isolated from biopsy sites of GVHD but not from normal tissue showed even greater numbers of tetraploid cells (mean = 78%, range: 15%-85%; N = 9) after culture. To mimic the inflammatory process in GVHD, allogeneic HLA-mismatched lymphocytes were mixed with normal keratinocytes. After 2 weeks, substantial numbers of aneuploid and tetraploid cells were evident in cultures with lymphocytes and with purified CD8 but not CD4 cells. Telomere length was substantially decreased in the lymphocyte-treated sample. No mutations were present in the p53 gene, although haploinsufficiency for p53 due to the loss of chromosome 17 was common in cells exposed to lymphocytes. These findings suggest that in GVHD, inflammation and repeated cell division correlate with the development of karyotypic abnormalities.

Fig. 1. Coculture of keratinocytes from healthy foreskins with allogeneic mis-matched lymphocytes

A) When mismatched allogeneic lymphocytes were co-cultured with foreskin keratinocytes for tetraploid cells could be observed in keratinocyte cultures co-cultured with allogeneic lymphocytes. An example of a FISH assay utilizing the X/Y probe is seen (left). Arrows point to tetraploid cells. Tetraploid cells are also distinguishable based on DNA content as measured by flow cytometric methods (right). B ) Keratinocytes cocultured with allogeneic were stained with FISH probes for chromosomes 7 and 8. Samples cultured with lymphocytes had significantly more tetraploidy and aneuploidy than samples expanded in the absence of lymphocytes. Tetraploidy appeared by day 3 of culture; aneuploidy was not seen until day 10.

Fig. 1. Coculture of keratinocytes from healthy foreskins with allogeneic mis-matched lymphocytes

A) When mismatched allogeneic lymphocytes were co-cultured with foreskin keratinocytes for tetraploid cells could be observed in keratinocyte cultures co-cultured with allogeneic lymphocytes. An example of a FISH assay utilizing the X/Y probe is seen (left). Arrows point to tetraploid cells. Tetraploid cells are also distinguishable based on DNA content as measured by flow cytometric methods (right). B ) Keratinocytes cocultured with allogeneic were stained with FISH probes for chromosomes 7 and 8. Samples cultured with lymphocytes had significantly more tetraploidy and aneuploidy than samples expanded in the absence of lymphocytes. Tetraploidy appeared by day 3 of culture; aneuploidy was not seen until day 10.

Fig. 2. Coculture of keratinocytes with CD8+ cells compared to CD4+ lymphocytes

A) Samples of PBLs from healthy donors were separated into CD8+ cells and CD4+ cells using magnetic columns and these were cultured with keratinocytes. Keratinocytes treated with allogeneic CD8+ cells developed the most tetraploidy and aneuploidy; cyclosporine blocked the effect of CD4+ and CD8+ cells. B) Following culture of keratinocytes with purified CD4 and CD8 cells, media was centrifuged to remove the cells and debris. Samples were then tested for cytokines using an immune-bead-based multiplex assay (Luminex). CD8 cells produced significantly more inflammatory cytokines than CD4 cells following co-culture with keratinocytes. Control samples consisted of those keratinocytes cultured for an equal time but without lymphocytes. The fold change compared to controls is graphed for each cytokine.

Fig. 3. Telomere lengths in keratinocytes cultured with lymphocytes

1. Left panel: Keratinocytes were cultured with PBLs as described in Methods and Materials and then passaged two times over the course of 17 days. Telomere length was then measured. PBLs caused a decrease in keratinocyte telomere, which was most prominent immediately following co-cultivation with lymphocytes.

2. Right panel: Keratinocytes were cultured with 30,000 purified CD4+, CD8+ and a mixture of similar numbers of CD4 and CD8 cells (i.e. 60,000 total lymphocytes), which were pre-incubated with cyclosporine. CD8+ cells showed the most dramatic change in telomere length.

Fig. 4. Loss of TP53 gene following coculture with keratinocytes

An aliquot of lymphocyte-treated keratinocytes was probed with locus specific p53 signal. Cells were probed with CEP 11 and LSP p53. When this was analyzed statistically using the Mann-Whitley test, an excess number of aneuploid and tetraploid cells showed either a gain or loss of p53 (p<0.001).