Sensitivity of leukemic T-cell lines to arsenic trioxide cytotoxicity is dependent on the induction of phosphatase B220/CD45R expression at the cell surface.

BACKGROUND: Arsenic trioxide (As2O3) is highly effective in treating acute promyelocytic leukemia (APL), but shows more variable therapeutic efficacy for other types of hematological malignancies. Previously, we reported that As2O3 selectively eliminates pathogenic B220-expressing T cells in autoimmune MRL/lpr mice. We investigated herein the relationship between As2O3 sensitivity of leukemic T-cell lines and the expression levels of the B220 isoform of transmembrane tyrosine phosphatase CD45. METHODS: GSH content, O2(-) production, and B220, HSP70, Fas and FasL membrane expression was measured by flow cytometry. Subcellular localization of B220 was determined by imaging flow cytometry. Cell death was analyzed by morphological changes, annexin V and propidium iodide staining, and caspase 8 and 9 activation. B220 mRNA expression was analyzed by RT-PCR. Activated NF-κB p50 was quantified by a DNA binding ELISA. RESULTS: We selected human (Jurkat, Jurkat variant J45.01, HPB-ALL) and mouse (EL-4, BW5147, L1210) T-cell lines for their marked differences in As2O3 sensitivity over a large range of doses (1 to 20 µM). Differences in redox status cannot explain the dramatic differences in As2O3 sensitivity observed among the T-cell lines. Unexpectedly, we found that B220 is differentially induced on As2O3-treated T-cell lines. As2O3 treatment for 24 h induced low (HPB-ALL), intermediate (Jurkat) and high (EL-4, BW5147) levels of B220 membrane expression, membrane-bound HSP70 and cell death, but inhibited NF-κB p50 nuclear translocation. When high levels of B220 expression were achieved with low doses of As2O3, the T-cell lines died by apoptosis only. When high doses of As2O3 were required to induce B220 expression, leukemic T cells died by both apoptosis and necrosis. CONCLUSIONS: Cellular redox status is not essential for As2O3 sensitivity of leukemic T cells, suggesting the existence of additional factors determining their sensitivity to As2O3 cytotoxicity. Phosphatase B220 could be such a factor of sensitivity. As2O3 treatment inhibits NF-κB p50 nuclear translocation, and induces B220 expression and cell death in a dose and time dependent manner. The levels of B220 induction on leukemic T cells strictly correlate with both the extent and form of cell death, B220 might therefore play a checkpoint role in death pathways.

P2X7 purinergic receptor plays a critical role in maintaining T-cell homeostasis and preventing lupus pathogenesis.

The severe lymphoproliferative and lupus diseases developed by MRL/lpr mice depend on interactions between the Fas lpr mutation and MRL genetic background. Thus, the Fas lpr mutation causes limited disease in C57BL/6 mice. We previously found that accumulating B220+ CD4-CD8- double negative (DN) T cells in MRL/lpr mice show defective P2X7 receptor ( P2X7)-induced cellular functions, suggesting that P2X7 contributes to T-cell homeostasis, along with Fas. Therefore, we generated a B6/lpr mouse strain (called B6/lpr-p2x7KO) carrying homozygous P2X7 knockout alleles. B6/lpr-p2x7KO mice accumulated high numbers of FasL-expressing B220+ DN T cells of CD45RBhighCD44high effector/memory CD8+ T-cell origin and developed severe lupus, characterized by leukocyte infiltration into the tissues, high levels of IgG anti-dsDNA and rheumatoid factor autoantibodies, and marked cytokine network dysregulation. B6/lpr-p2x7KO mice also exhibited a considerably reduced lifespan. P2X7 is therefore a novel regulator of T-cell homeostasis, of which cooperation with Fas is critical to prevent lymphoaccumulation and autoimmunity.

Variations in Cellular Responses of Mouse T Cells to Adenosine-5'-Triphosphate Stimulation Do Not Depend on P2X7 Receptor Expression Levels but on Their Activation and Differentiation Stage.

A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5'-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.

Low numbers of blood and salivary natural killer cells are associated with a better response to belimumab in primary Sjögren's syndrome: results of the BELISS study.

INTRODUCTION: In this study, we sought to address changes in blood lymphocyte subpopulations and labial salivary gland (LSG) inflammation after belimumab treatment in patients with primary Sjögren's syndrome (pSS) and to identify predictors of response to treatment. METHODS: Sequential blood lymphocyte subsets and LSG biopsies were analysed between week 0 (W0) and W28 in 15 patients with pSS treated with belimumab. Systemic response to treatment was defined as a decrease in the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index score of ≥3 points at W28. RESULTS: After belimumab, we observed a decrease in blood B lymphocytes primarily involving CD27-negative/immunoglobulin D-positive naïve B cells (p=0.008). Lymphocytic sialadenitis (focus score >1) that was present in 12 patients (80.0 %) before belimumab treatment became negative in 5 of them after treatment (p=0.03). The median (interquartile range) LSG B-cell/T-cell ratio decreased from 0.58 (0.5-0.67) to 0.50 (0.5-0.5) (p=0.06). B-cell activating factor (BAFF) staining was detected in 11 (78.6 %) of 14 patients before belimumab treatment compared with 7 (50.0 %) of 14 after belimumab therapy (p=0.10). The median percentage of BAFF-positive cells in foci significantly decreased from 27.5 % (10-40) to 5 % (0-20) (p=0.03). A systemic response was achieved in six patients (40 %). The only predictor of response was the presence of a low number of natural killer (NK) cells, both in blood (8.5 % [7-10] vs 11 % [9-21]; p=0.04) and in LSG (20.6/mm(3) [20.0-21.4] vs 30.0/mm(3) [25.0-100.0], p=0.003). Serum BAFF levels did not influence response to treatment. CONCLUSIONS: Low blood and salivary NK cell numbers are associated with a better response to belimumab. This suggests that two distinct subsets of pSS may exist: one with a predominant type I interferon (IFN)-BAFF-B-cell axis, representing good responders to belimumab; and one with a predominant type II IFN-NK cell axis, representing non-responders. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01160666 . Registered 9 July 2010.

Loss of P2X7 receptor plasma membrane expression and function in pathogenic B220+ double-negative T lymphocytes of autoimmune MRL/lpr mice.

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220(+) CD4(-)CD8(-) (DN) T lymphocytes. The precise ontogeny of B220(+) DN T cells is unknown. B220(+) DN T lymphocytes could be derived from effector CD4(+) and CD8(+) T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220(+) DN T-cell numbers in lymphoid organs. Importantly, we found that this B220(+) DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220(+) T cells observed in normal MRL(+/+) and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220(+) T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.