Simultaneous Cytokine Analysis by Cytometric Bead Array (CBA) Allows More Sensitive Detection of Leukemia-Reactive T Cells in Patients with Chronic Myeloid Leukemia.

Abstract

T cells recognizing truly leukemia-specific antigens like bcr/abl or other leukemia-associated antigens such as proteinase-3 and WT-1 are present in the T cell repertoire of leukemia patients and several studies have suggested that anti-leukemic T cells might be of clinical relevance. Detection of these leukemia-reactive T cells in CML patients is generally hampered by their low frequency making in vitro prestimulation necessary in most cases. Furthermore, shaping of the T cell repertoire by deletion of high affinity leukemia-reactive T cells and functional unresponsiveness are likely to have additional influence on T cell detection in structural or functional assays. Aim of our study was the detection of T cells specific for different CML-associated antigens chronic phase CML patients, most of them under imatinib treatment. In peripheral blood of CML patients we used a novel T cell assay (cytometric bead array = CBA), g-interferon ELISpot and tetramer staining in order to detect CML-reactive T cells against several known and new epitopes from bcr3/abl2, proteinase-3, c-abl and SOCS-2. By the standard g-interferon ELISpot peptide-specific T cells were not detectable in the vast majority of patients (33/34). In contrast peptide-specific cytokine release was detected in some of the patients by CBA upon pulsing with peptides from bcr3/abl2, proteinase-3, c-abl and SOCS-2. Peptide-specific cytokine release was detected particulary against a HLA-B8-restricted peptide from the fusion region of bcr3/abl2 (3/9 cases), against a HLA-A2-restricted epitope from proteinase-3 (3/11 cases), against a HLA-A2-restricted peptide from SOCS-2 (4/11 cases) and against two HLA-A2 and -B8-restricted epitopes from c-abl (3/11 and 2/9 cases respectively). T cell reactivity against the HLA-class-I epitopes from c-abl and SOCS-2 is described here for the first time. Interestingly, peptide-specific cytokine release was predominantly TNF-a, a significant IFN-g secretion was detected only in a few cases raising questions about the responding cells and their functional status. By tetramer staining low frequency T cells recognizing the bcr3/abl2 fusion protein were detected only in 2/15 patients, but after prestimulation of PBMC bcr/abl-specific T cells could be detected in 4/8 HLA-B8+ patients. Low T cell frequeny and deletion of high avidity T cells is a general obstacle for immune monitoring in cancer patients which may explain negative results obtained by g-IFN-ELISpot or tetramer staining. Furthermore, in CML an altered cytokine secretion pattern of T cells might be an additional limitation of functional T cell assays. Summarizing, we have found T cells recognizing CML-associated antigens which display a TNF-a-dominated cytokine secretion profile. This would have been missed using a standard g-IFN-ELISpot assay both because of the cytokine pattern of the T cells and the sensitivity of the assay. Using CBA there is a higher chance to detect low frequency leukemia-reactive T cells and to identify new immunotherapeutic targets in CML.

Vaccination with autologous non‐irradiated dendritic cells in patients with bcr/abl+ chronic myeloid leukaemia

“…T‐cell responses against CML‐associated peptides were detected in three patients (Patients 2, 3 and 4) and were directed against proteinase‐3, bcr3/abl2 and abl/bcr (Table IV). T‐cell responses against bcr/abl in CML patients have been described by several groups (Yotnda et al , 1998; Rezvani et al , 2003; Butt et al , 2004; Westermann et al , 2004, 2005; Gannagé et al , 2005). These data confirm that the fusion region of bcr3/abl2 contains immunogenic T‐cell epitopes, whereas immunogenic peptides from the bcr2/abl2 fusion region have not yet been clearly identified (Bocchia et al , 1995 and 1996).…”

“…In this patient, peptide‐specific cytokine secretion predominantly consisted of TNF‐ α , explaining negative γ ‐interferon ELISpot results. We have recently described an altered cytokine secretion profile with predominant TNF‐ α release in CML patients (Westermann et al , 2005). Gannagé et al (2005) also described a diminished interferon‐ γ accumulation in peptide‐specific T cells from CML patients.…”

“…Because of the paucity of PBMC, the supernatants for CBA in this study were taken from the ELISpot plates. A peptide‐specific response was defined as having a ratio ‘cytokine secretion of specific peptide: cytokine secretion of control peptide’≥2 (Westermann et al , 2005).…”

“…In recent decades, there has been an increasing evidence from both preclinical and clinical studies that the human T‐cell repertoire contains leukaemia‐reactive T cells, which might be of clinical relevance (Chen et al , 1992; Bocchia et al , 1996; Yotnda et al , 1998; Molldrem et al , 2000, 2003; Bellantuono et al , 2002; Westermann et al , 2004, 2005). Several leukaemia‐associated antigens, such as proteinase‐3 (and particularly its peptide PR‐1) (Molldrem et al , 2000), Wilms Tumor Protein‐1 (WT‐1) (Bellantuono et al , 2002) and bcr/abl (Chen et al , 1992; Bocchia et al , 1996), have been identified and may serve as target for T cells.…”

“…However, the same group described selective deletion of high‐affinity T cells from the T‐cell repertoire of CML patients, which may partially explain the failure of the immune system to eradicate the malignant cell clone (Molldrem et al , 2003). T‐cell responses against WT‐1‐ and bcr/abl have been described by several groups (Chen et al , 1992; Bocchia et al , 1996; Yotnda et al , 1998; Osman et al , 1999; Norbury et al , 2000; Clark & Christmas, 2001; Bellantuono et al , 2002; Westermann et al , 2004, 2005), but their activity in vivo remains a matter of debate. Clinical relevance of immunity against bcr/abl is also suggested by epidemiological data of Posthuma et al (1999), which showed that coexpression of human leucocyte antigen (HLA)‐A3 and –B8 (both HLA molecules can efficiently present immunogenic peptides from the bcr3/abl2 fusion region) reduces the risk of acquiring CML by approximately 50%.…”