Human CD4+ Memory T Cells Can Become CD4+IL-9+ T Cells

Background: IL-9 is a growth factor for T- and mast-cells that is secreted by human Th2 cells. We recently reported that IL-4+TGF-β directs mouse CD4+CD25−CD62L+ T cells to commit to inflammatory IL-9 producing CD4+ T cells. Methodology/Principal Findings: Here we show that human inducible regulatory T cells (iTregs) also express IL-9. IL-4+TGF-β induced higher levels of IL-9 expression in plate bound-anti-CD3 mAb (pbCD3)/soluble-anti-CD28 mAb (sCD28) activated human resting memory CD4+CD25−CD45RO+ T cells as compared to naïve CD4+CD25−CD45RA+ T cells. In addition, as compared to pbCD3/sCD28 plus TGF-β stimulation, IL-4+TGF-β stimulated memory CD4+CD25−CD45RO+ T cells expressed reduced FOXP3 protein. As analyzed by pre-amplification boosted single-cell real-time PCR, human CD4+IL-9+ T cells expressed GATA3 and RORC, but not IL-10, IL-13, IFNγ or IL-17A/F. Attempts to optimize IL-9 production by pbCD3/sCD28 and IL-4+TGF-β stimulated resting memory CD4+ T cells demonstrated that the addition of IL-1β, IL-12, and IL-21 further enhance IL-9 production. Conclusions/Significance: Taken together these data show both the differences and similarities between mouse and human CD4+IL9+ T cells and reaffirm the powerful influence of inflammatory cytokines to shape the response of activated CD4+ T cells to antigen

In Vivo Protection with Human Monoclonal Antibody S315 following Challenge with Diphtheria Toxin

Background: Morbidity and mortality from Corynebacterium diphtheriae is reduced by prompt administration of equine-derived diphtheria anti-toxin (DAT), which is in short supply worldwide. MassBiologics has developed a human monoclonal antibody (S315) to diphtheria toxin to provide a safer alternative to DAT and address critical supply issues. S315 prevents toxin binding to its putative host receptor and S315 pre-mixed with toxin increased survival in a guinea pig model of intoxication. To further evaluate the ability of S315 to provide in vivo protection, we established a post-exposure treatment model. Methods: Female Hartley guinea pigs (300-350g) were challenged subcutaneously with diphtheria toxin (0.03 to 0.09 Lf, limit of flocculation) to identify the minimum lethal dose. To evaluate anti-toxin efficacy, DAT or S315 was administered five hours post-toxin challenge and animals monitored for 30 days for signs of illness (lethargy, dehydration, weak limbs). Serum anti-diphtheria toxin antibodies were measured by ELISA and Vero cell toxin neutralization assays. Results: The minimum lethal toxin dose was 0.09 Lf. To determine the protective dose of DAT, 0.2 IU, 1.0 IU or 5.0 IU was administered intravenously post-toxin challenge (n=4/cohort). All 0.2 IU or 1.0 IU DAT-treated animals died, while one animal treated with 5.0 IU survived. DAT was subsequently evaluated at 5.0 IU, 10 IU, and 20 IU and compared to a cohort receiving 3.5 mg of S315. All untreated animals died within 72 hours and all antibody-treated animals survived. Dehydration was observed more frequently in the 5 IU and 10 IU DAT cohorts compared to the 20 IU and S315 cohorts. Conclusions: Treatment with S315 after diphtheria toxin exposure is protective; further studies will define a minimum effective dose of S315. This model mimics the route and timing of anti-toxin treatment in humans and provides a rigorous preclinical evaluation of a human antibody replacement for equine DAT

Biophysical Evaluation of Rhesus Macaque Fc Gamma Receptors Reveals Similar IgG Fc Glycoform Preferences to Human Receptors

Rhesus macaques are a common non-human primate model used in the evaluation of human monoclonal antibodies, molecules whose effector functions depend on a conserved N-linked glycan in the Fc region. This carbohydrate is a target of glycoengineering efforts aimed at altering antibody effector function by modulating the affinity of Fcγ receptors. For example, a reduction in the overall core fucose content is one such strategy that can increase antibody-mediated cellular cytotoxicity by increasing Fc-FcγRIIIa affinity. While the position of the Fc glycan is conserved in macaques, differences in the frequency of glycoforms and the use of an alternate monosaccharide in sialylated glycan species add a degree of uncertainty to the testing of glycoengineered human antibodies in rhesus macaques. Using a panel of 16 human IgG1 glycovariants, we measured the affinities of macaque FcγRs for differing glycoforms via surface plasmon resonance. Our results suggest that macaques are a tractable species in which to test the effects of antibody glycoengineering

Targeting chemoresistance and mitochondria-dependent metabolic reprogramming in acute myeloid leukemia

Chemoresistance often complicates the management of cancer, as noted in the instance of acute myeloid leukemia (AML). Mitochondrial function is considered important for the viability of AML blasts and appears to also modulate chemoresistance. As mitochondrial metabolism is aberrant in AML, any distinct pathways could be directly targeted to impact both cell viability and chemoresistance. Therefore, identifying and targeting those precise rogue elements of mitochondrial metabolism could be a valid therapeutic strategy in leukemia. Here, we review the evidence for abnormalities in mitochondria metabolic processes in AML cells, that likely impact chemoresistance. We further address several therapeutic approaches targeting isocitrate dehydrogenase 2 (IDH2), CD39, nicotinamide phosphoribosyl transferase (NAMPT), electron transport chain (ETC) complex in AML and also consider the roles of mesenchymal stromal cells. We propose the term “mitotherapy” to collectively refer to such regimens that attempt to override mitochondria-mediated metabolic reprogramming, as used by cancer cells. Mounting evidence suggests that mitotherapy could provide a complementary strategy to overcome chemoresistance in liquid cancers, as well as in solid tumors

Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression

The study of T regulatory cells (T reg cells) has been limited by the lack of specific surface markers and an inability to define mechanisms of suppression. We show that the expression of CD39/ENTPD1 in concert with CD73/ecto-5′-nucleotidase distinguishes CD4+/CD25+/Foxp3+ T reg cells from other T cells. These ectoenzymes generate pericellular adenosine from extracellular nucleotides. The coordinated expression of CD39/CD73 on T reg cells and the adenosine A2A receptor on activated T effector cells generates immunosuppressive loops, indicating roles in the inhibitory function of T reg cells. Consequently, T reg cells from Cd39-null mice show impaired suppressive properties in vitro and fail to block allograft rejection in vivo. We conclude that CD39 and CD73 are surface markers of T reg cells that impart a specific biochemical signature characterized by adenosine generation that has functional relevance for cellular immunoregulation

Activated mouse CD4+Foxp3−CD4^+Foxp3^− T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity

The regulatory activities of mouse $CD^4+Foxp3^+$ T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional $CD4^+Foxp3^−$ T cells in the periphery are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated $CD4^+Foxp3^−$ effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK-cell control of tumorigenesis is unclear. In the present study, we found that mitogen-activated $CD4^+Foxp3^−$ T cells exhibited potent suppressor function on NK-cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated $CD4^+Foxp3^−$ T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as administration of antibodies blocking either Qa-1 or NKG2A could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated $CD4^+Foxp3^−$ cells from Qa-1 knockout mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter regulated by the activation of adaptive immunity, a phenomenon we term as “activation-induced inhibition”. Thus, the regulatory role of activated $CD4^+Foxp3^−$ T cells in NK-cell activity must be taken into consideration in the future design of cancer therapies

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases