Myelodysplasia with Trisomy 8 Is Associated with a Cytotoxic CD8 T Cell Immune Response to Wilms Tumor-1 Protein (WT1).

Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated autoimmune process in some patients. Among 133 patients treated at NIH, predominantly with antithymocyte globulin, trisomy 8 as the sole karyotypic abnormality was specifically and significantly associated with favorable hematologic response to immunosuppresssion, as compared to patients with other cytogenetic abnormalities or with a normal karyotype. Trisomy 8 MDS patients show stable increases in the proportion of aneuploid bone marrow cells following immunosupppression (IST). We have reported that all of thirty patients with trisomy 8 had significant CD8 T cell expansion of one or more T cell receptor (TCR) Vβ subfamilies as measured by flow cytometry, and expanded subfamilies showed complimentary determining region 3 (CDR3) skewing by spectratyping. Sorted T cells of the expanded Vβ subfamilies, but none of the remaining subfamilies, specifically inhibit hematopoietic colony formation by trisomy 8 cell progenitors. Colony formation of cytogenetically normal cells from the same individuals was less or not inhibited. Here, we examined protein expression levels and measured the response of cytotoxic T cells to two antigens which we had found to be overexpressed in trisomy 8 CD34 cells by microarray analysis (Chen G et al, Blood In press): cyclin D1 (CD1) and Wilms tumor-1 antigen (WT1). Peripheral blood and bone marrow granulocytes of six trisomy 8 patients with refractory anemia all demonstrated WT1 expression levels 100–1000 x normal (N=38) by real time PCR; the three patients with normal cytogenetics had levels comparable to normal. Immunoblotting confirmed massively increased WT1 peptide in all six trisomy 8 patients tested, with five MDS patients of normal karyotype showing normal levels. Patient and control CD8 cells were cultured for 6 hours with WT1 and CD1-loaded HLA-A2-restricted antigen-presenting cells using three different peptides for WT1 and four for CD1. Cytotoxic CD8 T cells responses were identified by flow cytometric analysis of intracellular interferon-γ. Eight of 17 trisomy 8 patients showed significant responses (>8% CD8 cells activated compared to unstimulated samples; mean =5%) to WT1 but not to CD1 or non-peptide loaded antigen presenting cells. CD8 cell responses to WT1 measured using MHC class I A2-restricted tetramers were concordant with the results by intracellular staining in all 15 patients tested. In contrast, no responses to WT1 were seen in normal controls and five MDS patients lacking cytogenetic abnormalities. While PB of one of the three monosomy 7 patients showed upregulation of WT1, there was no cytotoxic lymphocyte response against the peptide; unlike trisomy 8 CD34 cells those from monosomy 7 patients did not express co-stimulatory molecules, HLA class I and B7.1. These data suggest an autoimmune pathophysiology for the cytopenia of trisomy 8 myelodysplasia with the following scenerio: WT1 is overexpressed by the trisomy 8 clone, resulting in a specific cytotoxic CD8 T cell immune response to WT1. Apoptotis of marrow cells results, either by cross reactivity or as a bystander effect. Improvement in hematopoiesis is seen following IST.