Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.

The impact of single amino acid substitutions in CD3gamma on the CD3epsilongamma interaction and T-cell receptor-CD3 complex formation.

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3gamma- and delta-dimers associate with the disulfide linked alphabeta- and zetazeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3gamma and CD3 in human T-cells. For this purpose, we have generated site-directed mutants of CD3gamma that were introduced in human T-cells defective in CD3gamma expression. Cell-surface and intracellular expression of the introduced CD3gamma chains was determined, as was the association with CD3delta, CD3, and the T-cell receptor. Although the introduction of wild type CD3gamma and CD3gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3gamma (82C-S), CD3gamma (85C-S), and CD3gamma (76Q-E) all resulted in an impaired association between CD3gamma and CD3 and a lack of cell-surface expressed CD3gamma. Finally, the introduction of CD3gamma (76Q-L) and CD3gamma (78Y-A) restored the expression of TCR-CD3deltagammazeta2 complexes, although the association between CD3gamma and CD3 was impaired. These results indicate that several amino acids in CD3gamma are essential for an optimal association between CD3gamma and CD3 and the assembly of a cell-surface expressed TCR-CD3deltagammazeta2 complex.

A novel and sensitive method for the detection of T cell stimulatory epitopes of alpha/beta- and gamma-gliadin.

BACKGROUND: It is now generally accepted that coeliac disease (CD) is caused by inflammatory T cell responses to gluten peptides bound to HLA-DQ2 or -DQ8 molecules. There is overwhelming evidence that CD patients can mount T cell responses to peptides found in both alpha-gliadin and gamma-gliadin molecules. Assays that would detect the presence or absence of such peptides in food would thus be accurate indicators of safety for consumption by CD patients. AIMS: The development of a sensitive method to detect T cell stimulatory epitopes of alpha-gliadin and gamma-gliadin molecules in food products. METHODS: Monoclonal antibodies (mAb) were raised against peptides encoding the T cell stimulatory epitopes of alpha-gliadin (amino acids (aa) 59-71) and aa gamma-gliadin (aa 142-153 and aa 147-159). These mAb competition assays were developed that quantitatively detect T cell stimulatory epitopes present on both intact proteins and peptides of sizes recognisable by CD4(+) T cells. RESULTS: With the mAb based competition assays, T cell epitopes were detected in pepsin/trypsin digests of wheat proteins and ethanol extracts of various food products, with detection levels lower than those reached with gluten specific T cells. Moreover, the presence of T cell stimulatory epitopes was also detected in preparations of barley, rye, and triticale, other cereals known to be toxic for CD patients. CONCLUSIONS: A new antibody based method has been developed, detecting the presence of T cell stimulatory gluten peptides. This can be used to further ensure the safety of food consumed by CD patients.

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells.

Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4+ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCL-specific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.