Reprogramming of immunosuppressive tumor microenvironment (TME) by targeting alternatively activated tumor associated macrophages (M2TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Tregs), represents a promising strategy for developing novel cancer immunotherapy. Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite and mediator of chronic inflammation, has emerged as a powerful immunosuppressor in the TME through engagement with one or more of its 4 receptors (EP1-EP4). We have developed E7046, an orally bioavailable EP4-specific antagonist and show here that E7046 has specific and potent inhibitory activity on PGE2-mediated pro-tumor myeloid cell differentiation and activation. E7046 treatment reduced the growth or even rejected established tumors in vivo in a manner dependent on both myeloid and CD8+ T cells. Furthermore, co-administration of E7046 and E7777, an IL-2-diphtheria toxin fusion protein that preferentially kills Tregs, synergistically disrupted the myeloid and Treg immunosuppressive networks, resulting in effective and durable anti-tumor immune responses in mouse tumor models. In the TME, E7046 and E7777 markedly increased ratios of CD8+granzymeB+ cytotoxic T cells (CTLs)/live Tregs and of M1-like/M2TAM, and converted a chronic inflammation phenotype into acute inflammation, shown by substantial induction of STAT1/IRF-1 and IFNγ-controlled genes. Notably, E7046 also showed synergistic anti-tumor activity when combined with anti-CTLA-4 antibodies, which have been reported to diminish intratumoral Tregs. Our studies thus reveal a specific myeloid cell differentiation-modifying activity by EP4 blockade and a novel combination of E7046 and E7777 as a means to synergistically mitigate both myeloid and Treg-derived immunosuppression for cancer treatment in preclinical models.

Heme oxygenase (HO) is one of the cytoprotective enzymes that can mitigate the effects of oxidative stress. Here, we found that the ho mRNA level oscillates in the brain of Drosophila melanogaster with two minima at the beginning of the day and night. This rhythm was partly masked by light as its pattern changed in constant darkness (DD). It followed a similar trend in the clock mutant per01 under light/dark regime (LD12:12); however, differences between time points were not statistically significant. In older flies (20 days old), the rhythm was vanished; however, 15 days of curcumin feeding restored this rhythm with an elevated ho mRNA level at all time points studied. In addition, flies exposed to paraquat had higher ho expression in the brain, but only at a specific time of the day which can be a protective response of the brain against stress. These findings suggest that the expression of ho in the fly's brain is regulated by the circadian clock, light, age, exposure to stress, and the presence of exogenous antioxidants. We also found that HO cross-talks with apoptosis and autophagy under different conditions. Induction of neuronal ho was accompanied by increased transcription of apoptosis and autophagy-related genes. However, this trend changed after exposure to curcumin and paraquat. Our results suggest that HO is involved in the control of apoptotic and autophagic key processes protecting the brain against oxidative damage.

Combinatorial strategies, such as targeting different immune checkpoint receptors, hold promise to increase the breadth and duration of the response to cancer therapy. Here we describe the preclinical evaluation of CTX-8371, a protein construct which combines PD-1 and PD-L1 targeting in one bispecific, tetravalent antibody. CTX-8371 matched or surpassed the activity of anti-PD-1 and PD-L1 benchmark antibodies in several in vitro T cell activation assays and outperformed clinically approved benchmarks in the subcutaneous MC38 colon and the B16F10 lung metastasis mouse tumor models. Investigation into the mechanism of action revealed that CTX-8371 co-engagement of PD-1 and PD-L1 induced the proteolytic cleavage and loss of cell surface PD-1, which is a novel and non-redundant mechanism that adds to the PD-1/PD-L1 signaling axis blockade. The combination of CTX-8371 and an agonistic anti-CD137 antibody further increased the anti-tumor efficacy with long-lasting curative therapeutic effect. In summary, CTX-8371 is a novel checkpoint inhibitor that might provide greater clinical benefit compared to current anti-PD-1 and PD-L1 antibodies, especially when combined with agents with orthogonal mechanisms of action, such as agonistic anti-CD137 antibodies.

CD8(+) T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8(+) T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8(+) T cells, CD8 coreceptor and Plcgamma1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8(+) T cells through association with specific enhancers. Short-lived Klrg1(hi)CD127(lo) effector CD8(+) T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.

A single adenosine receptor gene (dAdoR) has been detected in Drosophila melanogaster. However, its function in different cell types of the nervous system is mostly unknown. Therefore, we overexpressed or silenced the dAdoR gene in eye photoreceptors, all neurons, or glial cells and examined the fitness of flies, the amount and daily pattern of sleep, and the influence of dAdoR silencing on Bruchpilot (BRP) presynaptic protein. Furthermore, we examined the dAdoR and brp gene expression in young and old flies. We found that a higher level of dAdoR in the retina photoreceptors, all neurons, and glial cells negatively influenced the survival rate and lifespan of male and female Drosophila in a cell-dependent manner and to a different extent depending on the age of the flies. In old flies, expression of both dAdoR and brp was higher than in young ones. An excess of dAdoR in neurons improved climbing in older individuals. It also influenced sleep by lengthening nighttime sleep and siesta. In turn, silencing of dAdoR decreased the lifespan of flies, although it increased the survival rate of young flies. It hindered the climbing of older males and females, but did not change sleep. Silencing also affected the daily pattern of BRP abundance, especially when dAdoR expression was decreased in glial cells. The obtained results indicate the role of adenosine and dAdoR in the regulation of fitness in flies that is based on communication between neurons and glial cells, and the effect of glial cells on synapses.

Dysregulated CD4(+) T cell responses and alterations in T regulatory cells (T(reg) cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in T(reg) cells causes IBD because of proinflammatory cytokine-producing CD4(+) T cells infiltrating the colon. Provision of WT T(reg) cells prevented IBD, demonstrating that alterations in T(reg) cells are responsible for the disease. Furthermore, Bcl11b-deficient T(reg) cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced T(reg) cells (iT(reg) cells) after Tgf-β treatment of conventional CD4(+) T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for T(reg) suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4(+) T cells in response to Tgf-β and generation of iT(reg) cells.

Transcriptional control of gene expression in double-positive (DP) thymocytes remains poorly understood. We show that the transcription factor BCL11B plays a critical role in DP thymocytes by controlling positive selection of both CD4 and CD8 lineages. BCL11B-deficient DP thymocytes rearrange T cell receptor (TCR) alpha; however, they display impaired proximal TCR signaling and attenuated extracellular signal-regulated kinase phosphorylation and calcium flux, which are all required for initiation of positive selection. Further, provision of transgenic TCRs did not improve positive selection of BCL11B-deficient DP thymocytes. BCL11B-deficient DP thymocytes have altered expression of genes with a role in positive selection, TCR signaling, and other signaling pathways intersecting the TCR, which may account for the defect. BCL11B-deficient DP thymocytes also presented increased susceptibility to spontaneous apoptosis associated with high levels of cleaved caspase-3 and an altered balance of proapoptotic/prosurvival factors. This latter susceptibility was manifested even in the absence of TCR signaling and was only partially rescued by provision of the BCL2 transgene, indicating that control of DP thymocyte survival by BCL11B is nonredundant and, at least in part, independent of BCL2 prosurvival factors.