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

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

1. a novel T cell assay (cytometric bead array = CBA),

2. g-interferon ELISpot and

3. 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.