Targeting the Ataxia Telangiectasia Mutated-null Phenotype in Chronic Lymphocytic Leukemia with Pro-oxidants

Inactivation of the Ataxia Telangiectasia Mutated gene in chronic lymphocytic leukemia results in resistance to p53-dependent apoptosis and inferior responses to treatment with DNA damaging agents. Hence, p53-independent strategies are required to target Ataxia Telangiectasia Mutated-deficient chronic lymphocytic leukemia. As Ataxia Telangiectasia Mutated has been implicated in redox homeostasis, we investigated the effect of the Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia genotype on cellular responses to oxidative stress with a view to therapeutic targeting. We found that in comparison to Ataxia Telangiectasia Mutated-wild type chronic lymphocytic leukemia, pro-oxidant treatment of Ataxia Telangiectasia Mutated-null cells led to reduced binding of NF-E2 p45-related factor-2 to antioxidant response elements and thus decreased expression of target genes. Furthermore, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia cells contained lower levels of antioxidants and elevated mitochondrial reactive oxygen species. Consequently, Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia, but not tumours with 11q deletion or TP53 mutations, exhibited differentially increased sensitivity to pro-oxidants both in vitro and in vivo. We found that cell death was mediated by a p53- and caspase-independent mechanism associated with apoptosis inducing factor activity. Together, these data suggest that defective redox-homeostasis represents an attractive therapeutic target for Ataxia Telangiectasia Mutated-null chronic lymphocytic leukemia

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-w (IFN-w) (13 patients), against the 13 types of IFN-a (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men

Over-expression of CD200 in Acute Myeloid Leukemia Mediates the Expansion of Regulatory T-lymphocytes and Directly Inhibits Natural Killer Cell Tumor Immunity

CD200 is a type-1 transmembrane glycoprotein which suppresses inflammatory and autoimmune responses by signalling through its cognate transmembrane receptor homologue (CD200R). Normally, CD200 expression is restricted to immune privileged sites where it enhances immune tolerance through mechanisms that include modulating the expansion of FOXP3+ regulatory T-lymphocytes (T-regs) and suppressing macrophage cytolytic activity. Furthermore, leukocyte associated CD200 has been reported to suppress Natural Killer (NK) cell activity in vivo. Pathologically, we have previously shown that CD200 over-expression on leukemic blasts in around 50% of acute myeloid leukemia (AML) patients is significantly associated with a poor overall survival (Tonks et al, Leukemia, 2007). Given the existing evidence that T-reg frequency and NK cell function influence blast clearance and long-term survival in AML, we investigated the possibility that CD200 expression in AML may be directly suppressing anti-tumor immunity in this disease. Here we present evidence that CD200+ AML can suppress host anti-tumor responses by augmenting the frequency of AML patient T-regs and by direct inhibition of NK cell anti-tumor activity. We also show that targeting the interaction between CD200 and its receptor might provide a new strategy for the treatment of AML. Bone marrow aspirates from 91 diagnostic AML patients were analysed by multiparameter flow cytometry for blast CD200 protein expression. We found that the level of blast CD200 expression directly correlated with an increased frequency of T-regs (CD4+CD25++FoxP3+; R=0.78, p=0.0008). Measuring 3H-thymidine incorporation, we show that T-regs isolated from AML patients by MACS® separation inhibited T-cell proliferation (induced by CD3 and CD28 stimulation) at ratios <0.1%, thus confirming that patients T-regs were functional. In contrast to T-regs, NK cell frequency (CD45+CD19−CD3−CD56+) did not correlate with the level of AML blast CD200 expression (R=0.15, p=0.851), however, NK cell subpopulation bivariate analysis using CD56 and CD16 demonstrated that the CD56dimCD16+ (the principle active NK population) was significantly reduced by over 50% in CD200+ AML patients (36±5% compared to 15±5%, p=0.009). Furthermore, CD200 expression on target cells appeared to have a direct effect on the cytotoxic activity of NK cells; co-culture of NK cells with CD200+ targets resulted in decreased CD107a expression (a marker for cytolytic granules) in NK cells (23±4% vs 12±5%, p=0.038) and decreased apoptosis of the target cells (19±1% vs 10±1%, p=0.041). Since CD200R was detected on NK cells in AML patients, it was likely that CD200 was having a direct effect on suppression of NK cytotoxicity. This was supported by the significant recovery of NK cytolytic activity against CD200+ blasts in the presence of a CD200 blocking antibody (5±1% vs 11±2% CD107a+ NK cells, p=0.046) whereas there was no change seen with CD200− blasts (19±4% vs 19±3%). In conclusion, these findings suggest that CD200 expression on leukemic blasts plays an influential role in suppressing anti-tumor immunity in AML patients through modulating the expansion of functionally suppressive T-regs and directly suppressing NK cell cytolytic activity. In this study blocking CD200 interaction with its receptor was able to recover a significant proportion of patient NK activity, making CD200 a potential therapeutic target for CD200+ AML

CD200 Inhibits Memory Th1 Cell Function in Acute Myeloid Leukaemia (AML)

CD200 is a cell-surface glycoprotein that is normally expressed in tissues of the immune system, where its role is to protect immune privileged sites. We previously established CD200 to be frequently over-expressed and associated with poor AML patient outcome. In this study, we investigated the possibility that CD200 expression may mediate suppression of T-cell function in this disease. Using multiparameter flow cytometry, we compared PMA/ionomycin stimulated CD8+ T-cell cytotoxic potential (CD107a expression) and the frequency of intracellular TNFa, IL-2 and IFNc producing CD4+/CD8+ memory T-cells between CD200hi and CD200lo patients. We demonstrated that both the magnitude of the CD8+ memory cytotoxic T-cell response and the Th1 cytokine producing CD4+ memory helper T-cells was significantly inhibited in CD200hi AML patients (P < 0.05). Further, using ELISPOT assays to measure IFNg release we showed that the Th1 memory response to common viral antigens was significantly reduced by 75% in CD200hi versus CD200lo AML patients(P < 0.05). Recovery of IFNc release in response to recall antigens was observed in CD4+ memory T-cells incubated with a blocking antibody to CD200R. In conclusion, this study shows a correlation between T-cell dysfunction and expression of CD200 which suggests targeting this axis could be therapeutically beneficial for AML CD200hi patients

Intracellular Thiol Oxidation Is Linked with Loss of Δψm and Disease Progression in Acute Promyelocytic Leukaemia

Acute promyelocytic leukaemia (APL) is a type of myeloid malignancy defined by the chromosomal translocation t(15;17) and subsequent expression of the PML-RARα fusion protein. Long term remission in APL is achieved through a combination of high dose all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the latter of which has potential to induce mitochondrial derived oxidative stress and initiate the intrinsic apoptosis pathway. ATO is particularly effective in the treatment of APL when compared with acute myeloid leukaemia (AML), suggesting that the mitochondrial membrane potential (Δψm) of APL cells is compromised. Here we show that loss of Δψm is associated with intracellular thiol oxidation and disease progression in APL. The mitochondrial probe 3,3'-diethyloxacrbocyanine iodide (DiOC2(3)) was used to evaluate MRP8-PML/RARA transgenic mice Δψm in bone marrow mononuclear cells (BMMC) at three disease stages: 1) pre-APL, 2) APL and 3) ATRA treated APL. Our data show that MRP8-PML/RARA BMMC cells during the APL stage accumulate significantly less DiOC2(3) compared with pre-APL cells (0.05±0.02 vs 0.24±0.05, p0.05). This finding indicates a loss of Δψm in APL. This may result in free radical leakage from the mitochondria, which in turn could oxidise intracellular thiols. To evaluate intracellular thiol oxidation, a previously optimised flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide (F5M) was used. Our data show a significant decrease in F5M MFI for MRP8-PML/RARA BMMC cells during the APL stage, compared with pre-APL cells (219.01±47.67 vs 672.66±131.04, p<0.05; data are F5M MFI relative to unstained controls). This finding signifies that there is more thiol oxidation at the APL stage. ATRA treated APL mice showed an equivalent F5M MFI to that of pre-APL cells. Confocal microscopy confirmed that the F5M signal was intracellular. This observation shows that there is an increase in intracellular thiol oxidation in APL, which may be tracked during disease progression and treatment course. This finding may explain why APL cells are more sensitive to pro-oxidant treatments i.e. ATO. To understand these observations further, the APL cell line (NB4) was subjected to glucose oxidase (GOX) mediated oxidative stress in cell culture over a 4 hour time-course. F5M MFI signal was compared with the THP1 and Kasumi-1 AML cell lines subjected to the same treatment. All cell lines showed similar significant increases in F5M MFI after 1 hour GOX treatment relative to control (p0.05), whereas F5M MFI Kasumi-1 remained significantly increased after 4 hours (p<0.05) relative to control. This finding indicates differences in the sensitivity to oxidative stress between APL and AML cells. Taken together, this study shows that APL cells are more sensitive to oxidative stress compared with AML cells. Our F5M flow cytometric assay illustrates that an increase in cellular thiol oxidation is linked to disease progression in APL, with loss of Δψm associated with this process. Our F5M flow cytometric assay may therefore have clinical utility in monitoring treatment efficacy in APL

CD200 and PD1-L1 in AML Are Associated with Expanded PD-1+ Late Differentiated CD8+ T Cells and a Decreased CD4:CD8 Ratio: a New Link Between Distinct Immunosuppressive Pathways

Long-term remission for acute myeloid leukemia (AML) is still not achieved for the majority of patients and consequently there is a need for new treatments to consolidate current therapy. A promising approach is to augment the anti-tumor immune response in these patients; however most cancers do not activate immune effector cells because they express immunosuppressive ligands. Previously we showed that CD200 overexpression on AML blasts suppresses memory CD4+ and CD8+ T cell effector function through engagement with CD200 receptor (CD200R) on these cells. Blocking CD200:CD200R, however, only partially restored T cell activity, suggesting that alternative immunosuppressive mechanisms were involved. Recently, promising clinical outcomes have been reported for melanoma and non-small cell lung cancer using humanized antibodies targeting another immunosuppressive receptor, PD-1, and we therefore investigated whether this could be contributing to the immunosuppression of T cell effector responses in CD200hi AML patients. Initially, we investigated whether CD200 and the immunosuppressive ligand for PD-1, PD1-L1, were co-expressed in AML blasts at diagnosis. Affymetrix gene expression data from 158 AML blasts showed that AML patients in the upper quartile for CD200 expression (CD200hi) had 10-fold higher levels of PD1-L1 expression compared to CD200lo (lower quartile) patients. Analysis of CD200 and PD1-L1 protein expression on AML blast cells confirmed this association at the protein level (r2 = 0.49; p50%) reduction in the frequency of activated 7E7 T cells; however, when both CD200 and PD1-L1 were co-expressed, T cell activation was almost ablated (~90% reduction; p<0.01). Moreover, the strength of the TNFα response was also reduced in co-culture assays where either CD200 or PD1-L1 were present, indicating a direct effect at the level of CD8+ T cell function (2.8 ± 0.5 vs 1.7 ± 0.5; p<0.05). These data demonstrate that CD200:CD200R and PD1-L1:PD-1 engagement on T cells can act in tandem to augment immunosuppression of CD8+ T cells. In summary, we show for the first time that the immunosuppressive molecules, CD200 and PD1-L1 appear to be co-regulated on AML blasts and that these can act in combination to profoundly suppress T cell activation. Further, we show that CD200:CD200R engagement induces PD-1+ CD8+ T cells. Taken together we propose a novel CD200/PD1-L1 immunotherapeutic synapse in AML which should be targeted by combining CD200:CD200R and PD1-L1:PD-1 blockade in immunotherapy of AML

An Ambulatory Pediatric Unit

Description and discussion of organization, development and responses of parents to a newly created facility for ambulatory pediatric care.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66646/2/10.1177_000992287000900805.pd