Gene profiling studies of patients with chronic lymphocytic leukemia (CLL) has revealed increased expression of Ror1, a cell surface receptor tyrosine kinase. The aim of present study was to analyze gene and protein expression of Ror1 in CLL cells and normal blood leukocytes. Gene expression analysis reverse transcriptionpolymerase chain reaction of ROR1 revealed that all patients with CLL (n 5 100) spontaneously expressed ROR1 mRNA whereas enriched blood B and T cells as well as granulocytes from healthy donors (n 5 10) were negative. A strong nonphysiological activation signal (PMA/ionomycin) was required to induce expression in vitro in normal lymphocytes. Major genomic aberrations (mutations or truncation) of ROR1 were not observed. Protein expression was analyzed by Western blot using a panel of polyclonal anti-Ror antibodies as well as flow cytometry. Blood lymphocytes from 18/18 CLL patients, but none of the 10 healthy donors, expressed surface Ror1. The majority of CLL cells exhibited Ror1 surface expression (71% mean; range 36-92%) with a mean fluorescence intensity (MFI) of 20 (range 10-45). The corresponding MFI of CD19 on CLL cells was 26 (range 9-48). There was no difference in the Ror1 protein expression comparing IgVH mutated and unmutated cases as well as progressive and nonprogressive CLL patients. Two different variants of the Ror1 protein, 105 and 130 kDa, were identified. The Ror1 protein expression in patients with CLL but not in normal leukocytes merits further studies of its role in the pathobiology of CLL, which may provide a basis for development of Ror1 directed targeted therapy. ' 2008 Wiley-Liss, Inc. Key words: B-CLL; tyrosine kinase receptors; Ror1Chronic lymphocytic leukemia (CLL) originates from B lymphocytes, which differ in activation and maturation stage and are derived from antigen experienced B cells with different immunoglobulin heavy chain variable (IgVH) gene mutations. 1 Patients with mutated IgVH genes have a better prognosis compared to patients with unmutated genes. 2,3 Global gene expression profiling studies have revealed partly distinguishing but in general overlapping expression profiles in mutated and unmutated leukemic B cells, suggesting a common phenotype. 4,5 Gene expression profiling studies showed a 43.8-fold increase of the orphan receptor tyrosine kinase (RTK) ROR1 in CLL cells. 4 Ror1 is a member of the RTK family of orphan receptors related to muscle specific kinase and Trk neurotrophin receptors. [6][7][8] Ror receptors are cell surface receptors participating in signal transduction, cell-cell interaction, regulation of cell proliferation, differentiation, cell metabolism and survival. 7,9 They are evolutionarily highly conserved between different species e.g., human, mouse, Drosophila and C. elegans, suggesting important biological functions.The human ROR1 gene has a coding region of 2814 bp with a predicted 937 amino acids sequence and 105 kDa protein size including an Ig-like domain, cysteine-rich domain, kringle domain, tyrosine kinase domain and p...
Analysis of cytokine production is a tool to functionally characterise T cells. In this study, spontaneous and polyclonal activation induced cytokine production in T cells were assessed by flow cytometry in patients with B-CLL. Patients with progressive disease had a significantly increased number of T cells spontaneously producing IL-2, IL-4 and GM-CSF as compared to healthy donors and patients with non-progressive CLL, which was not the case for TNF-alpha and IFN-gamma producing T cells. However, no difference in the frequency of T cells producing these cytokines was seen comparing patients with non-progressive disease to control donors. Polyclonal activation of B-CLL T cells in vitro induced an increased proportion of T cells producing these five cytokines in patients as well as in control donors, indicating that T cells in CLL patients might have a relatively well preserved functional capacity. However, the increase in GM-CSF, TNF-alpha and IL-4 producing T cells was more marked in CLL patients than in controls. Furthermore, following activation, a higher frequency of cytokine-producing T cells was noted in patients with progressive disease as compared to those with non-progressive disease. The augmented number of cytokine-producing T cells in CLL may indicate an up-regulated capability of T cells to secrete cytokines, especially in patients with progressive CLL. The increased production of the T cell derived cytokines GM-CSF, TNF-alpha, IL-4 and IL-2 is interesting, as these cytokines have previously been shown to support growth of B-CLL leukaemic cells in vitro and as T cells might specifically recognise the autologous leukaemic B cells in vivo. The findings may suggest a role for T cells in the pathogenesis of B-CLL.
Summary. This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by realtime reverse transcription polymerase chain reaction (RT-PCR) revealed that granulocyte±macrophage colony-stimulating factor (GM-CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were ginterferon (IFN), interleukin (IL)-2 and tumour necrosis factor (TNF)-a, but not IL-4. This profile suggests a type 1 anti-B-CLL T-cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T-cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T-cell DNA synthesis proliferation. The proliferative T-cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T-cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T-cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti-CLL T-cell immunity is warranted that may facilitate the development of effective anti-tumour vaccines in CLL.
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