Watch your clock: it matters for immunotherapy.

Does time of day matter for cancer immunotherapy? Whereas the concept of optimizing the time of treatment is well documented for chemotherapy, whether it applies to immunotherapy, a revolutionizing treatment exploiting the power of immune cells to control tumors, has recently been addressed in a study published in Cell.

Regulation of Cytotoxic CD8+ T Cells by the Circadian Clock.

Most aspects of physiology, including immunity, present 24-h variations called circadian rhythms. In this review, we examine the literature on the circadian regulation of CD8+ T cells, which are important to fight intracellular infections and tumors. CD8+ T cells express circadian clock genes, and ∼6% of their transcriptome presents circadian oscillations. CD8+ T cell counts present 24-h rhythms in the blood and in secondary lymphoid organs, which depend on the clock in these cells as well as on hormonal rhythms. Moreover, the strength of the response of these cells to Ag presentation varies according to time of day, a rhythm dependent on the CD8+ T cell clock. The relevance of CD8+ T cell circadian rhythms is shown by the daily variations in the fight of intracellular infections. Such a circadian regulation also has implications for cancer, as well as the optimization of vaccination and immunotherapy.

Role of NR4A family members in myeloid cells and leukemia.

The myeloid cellular compartment comprises monocytes, dendritic cells (DCs), macrophages and granulocytes. As diverse as this group of cells may be, they are all an important part of the innate immune system and are therefore linked by the necessity to be acutely sensitive to their environment and to rapidly and appropriately respond to any changes that may occur. The nuclear orphan receptors NR4A1, NR4A2 and NR4A3 are encoded by immediate early genes as their expression is rapidly induced in response to various signals. It is perhaps because of this characteristic that this family of transcription factors has many known roles in myeloid cells. In this review, we will regroup and discuss the diverse roles NR4As have in different myeloid cell subsets, including in differentiation, migration, activation, and metabolism. We will also highlight the importance these molecules have in the development of myeloid leukemia.

p16INK4a Regulates Cellular Senescence in PD-1-Expressing Human T Cells.

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

Roles and mechanisms of BAP1 deubiquitinase in tumor suppression.

The BAP1 gene has emerged as a major tumor suppressor mutated with various frequencies in numerous human malignancies, including uveal melanoma, malignant pleural mesothelioma, clear cell renal cell carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, and thymic epithelial tumors. BAP1 mutations are also observed at low frequency in other malignancies including breast, colorectal, pancreatic, and bladder cancers. BAP1 germline mutations are associated with high incidence of mesothelioma, uveal melanoma, and other cancers, defining the "BAP1 cancer syndrome." Interestingly, germline BAP1 mutations constitute an important paradigm for gene-environment interactions, as loss of BAP1 predisposes to carcinogen-induced tumorigenesis. Inactivating mutations of BAP1 are also identified in sporadic cancers, denoting the importance of this gene for normal tissue homeostasis and tumor suppression, although some oncogenic properties have also been attributed to BAP1. BAP1 belongs to the deubiquitinase superfamily of enzymes, which are responsible for the maturation and turnover of ubiquitin as well as the reversal of substrate ubiquitination, thus regulating ubiquitin signaling. BAP1 is predominantly nuclear and interacts with several chromatin-associated factors, assembling multi-protein complexes with mutually exclusive partners. BAP1 exerts its function through highly regulated deubiquitination of its substrates. As such, BAP1 orchestrates chromatin-associated processes including gene expression, DNA replication, and DNA repair. BAP1 also exerts cytoplasmic functions, notably in regulating Ca2+ signaling at the endoplasmic reticulum. This DUB is also subjected to multiple post-translational modifications, notably phosphorylation and ubiquitination, indicating that several signaling pathways tightly regulate its function. Recent progress indicated that BAP1 plays essential roles in multiple cellular processes including cell proliferation and differentiation, cell metabolism, as well as cell survival and death. In this review, we summarize the biological and molecular functions of BAP1 and explain how the inactivation of this DUB might cause human cancers. We also highlight some of the unresolved questions and suggest potential new directions.

GCNT1-Mediated O-Glycosylation of the Sialomucin CD43 Is a Sensitive Indicator of Notch Signaling in Activated T Cells.

Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.

Role of the Orphan Nuclear Receptor NR4A Family in T-Cell Biology.

The nuclear orphan receptors NR4A1, NR4A2, and NR4A3 are immediate early genes that are induced by various signals. They act as transcription factors and their activity is not regulated by ligand binding and are thus regulated via their expression levels. Their expression is transiently induced in T cells by triggering of the T cell receptor following antigen recognition during both thymic differentiation and peripheral T cell responses. In this review, we will discuss how NR4A family members impact different aspects of the life of a T cell from thymic differentiation to peripheral response against infections and cancer.

Enhanced myelopoiesis and aggravated arthritis in S100a8-deficient mice.

Expressed strongly by myeloid cells, damage-associated molecular pattern (DAMP) proteins S100A8 and S100A9 are found in the serum of patients with infectious and autoimmune diseases. Compared to S100A9, the role of S100A8 is controversial. We investigated its biological activity in collagen-induced arthritis using the first known viable and fertile S100a8-deficient (S100a8-/-) mouse. Although comparable to the wild type (WT) in terms of lymphocyte distribution in blood and in the primary and secondary lymphoid organs, S100a8-/- mice had increased numbers of neutrophils, monocytes and dendritic cells in the blood and bone marrow, and these all expressed myeloid markers such as CD11b, Ly6G and CD86 more strongly. Granulocyte-macrophage common precursors were increased in S100a8-/- bone marrow and yielded greater numbers of macrophages and dendritic cells in culture. The animals also developed more severe arthritic disease leading to aggravated osteoclast activity and bone destruction. These findings were correlated with increased inflammatory cell infiltration and cytokine secretion in the paws. This study suggests that S100A8 is an anti-inflammatory DAMP that regulates myeloid cell differentiation, thereby mitigating the development of experimental arthritis.

The orphan nuclear receptor NR4A3 controls the differentiation of monocyte-derived dendritic cells following microbial stimulation.

In response to microbial stimulation, monocytes can differentiate into macrophages or monocyte-derived dendritic cells (MoDCs) but the molecular requirements guiding these possible fates are poorly understood. In addition, the physiological importance of MoDCs in the host cellular and immune responses to microbes remains elusive. Here, we demonstrate that the nuclear orphan receptor NR4A3 is required for the proper differentiation of MoDCs but not for other types of DCs. Indeed, the generation of DC-SIGN+ MoDCs in response to LPS was severely impaired in Nr4a3-/- mice, which resulted in the inability to mount optimal CD8+ T cell responses to gram-negative bacteria. Transcriptomic analyses revealed that NR4A3 is required to skew monocyte differentiation toward MoDCs, at the expense of macrophages, and allows the acquisition of migratory characteristics required for MoDC function. Altogether, our data identify that the NR4A3 transcription factor is required to guide the fate of monocytes toward MoDCs.

Early Notch Signals Induce a Pathogenic Molecular Signature during Priming of Alloantigen-Specific Conventional CD4+ T Cells in Graft-versus-Host Disease.

Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 h after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit the expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv versus Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection as GVHD severity was similar in the recipients of wild-type Tconv combined with Notch-deprived versus wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4+ Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and Th cell polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with the pathogenic effects of Notch in T cells at the earliest stages of GVHD.

The Notch signaling pathway controls CD8+ T cell differentiation independently of the classical effector HES1.

During CD8+ T cell response, Notch signaling controls short-lived-effector-cell (SLEC) generation, but the exact mechanisms by which it does so remains unclear. The Notch signaling pathway can act as a key regulator of Akt signaling via direct transcriptional induction of Hes1, which will then repress the transcription of Pten, an inhibitor of Akt signaling. As both Notch and Akt signaling can promote effector CD8+ T cell differentiation, we asked whether Notch signaling influences SLEC differentiation via the HES1-PTEN axis. Here, we demonstrate that HES1 deficiency in murine CD8+ T cells did not impact SLEC differentiation. Moreover, we show that Pten transcriptional repression in effector CD8+ T cells is not mediated by Notch signaling although Akt activation requires Notch signaling. Therefore, HES1 is not an effector of Notch signaling during CD8+ T cell response.

Neuropilin-1 expression in adipose tissue macrophages protects against obesity and metabolic syndrome.

Obesity gives rise to metabolic complications by mechanisms that are poorly understood. Although chronic inflammatory signaling in adipose tissue is typically associated with metabolic deficiencies linked to excessive weight gain, we identified a subset of neuropilin-1 (NRP1)-expressing myeloid cells that accumulate in adipose tissue and protect against obesity and metabolic syndrome. Ablation of NRP1 in macrophages compromised lipid uptake in these cells, which reduced substrates for fatty acid ß-oxidation and shifted energy metabolism of these macrophages toward a more inflammatory glycolytic metabolism. Conditional deletion of NRP1 in LysM Cre-expressing cells leads to inadequate adipose vascularization, accelerated weight gain, and reduced insulin sensitivity even independent of weight gain. Transfer of NRP1+ hematopoietic cells improved glucose homeostasis, resulting in the reversal of a prediabetic phenotype. Our findings suggest a pivotal role for adipose tissue-resident NRP1+-expressing macrophages in driving healthy weight gain and maintaining glucose tolerance.

The circadian clock in immune cells controls the magnitude of Leishmania parasite infection.

The intracellular parasite Leishmania uses neutrophils and macrophages as host cells upon infection. These immune cells harbour their own intrinsic circadian clocks, known to influence many aspects of their functions. Therefore, we tested whether the host circadian clocks regulate the magnitude of Leishmania major infection in mice. The extent of parasitic infection varied over 24 h in bone marrow-derived macrophages in vitro and in two different in vivo models, footpad and peritoneal cavity infection. In vivo this was paralleled by time of day-dependent neutrophil and macrophage infiltration to the infection site and rhythmic chemokine expression. Thus, rhythmic parasitic infection observed in vivo was likely initiated by the circadian expression of chemoattractants and the subsequent rhythmic infiltration of neutrophils and macrophages. Importantly, all rhythms were abolished in clock-deficient macrophages and when mice lacking the circadian clock in immune cells were infected. Therefore we demonstrated a critical role for the circadian clocks in immune cells in modulating the magnitude of Leishmania infection. To our knowledge this is the first report showing that the circadian clock controls infection by protozoan parasites in mammals. Understanding the timed regulation of host-parasite interactions will allow developing better prophylactic and therapeutic strategies to fight off vector-borne diseases.

Enhancing circadian clock function in cancer cells inhibits tumor growth.

BACKGROUND: Circadian clocks control cell cycle factors, and circadian disruption promotes cancer. To address whether enhancing circadian rhythmicity in tumor cells affects cell cycle progression and reduces proliferation, we compared growth and cell cycle events of B16 melanoma cells and tumors with either a functional or dysfunctional clock. RESULTS: We found that clock genes were suppressed in B16 cells and tumors, but treatments inducing circadian rhythmicity, such as dexamethasone, forskolin and heat shock, triggered rhythmic clock and cell cycle gene expression, which resulted in fewer cells in S phase and more in G1 phase. Accordingly, B16 proliferation in vitro and tumor growth in vivo was slowed down. Similar effects were observed in human colon carcinoma HCT-116 cells. Notably, the effects of dexamethasone were not due to an increase in apoptosis nor to an enhancement of immune cell recruitment to the tumor. Knocking down the essential clock gene Bmal1 in B16 tumors prevented the effects of dexamethasone on tumor growth and cell cycle events. CONCLUSIONS: Here we demonstrated that the effects of dexamethasone on cell cycle and tumor growth are mediated by the tumor-intrinsic circadian clock. Thus, our work reveals that enhancing circadian clock function might represent a novel strategy to control cancer progression.

Inflammation enhances the vaccination potential of CD40-activated B cells in mice.

Vaccination with antigen-pulsed CD40-activated B (CD40-B) cells can efficiently lead to the in vivo differentiation of naive CD8+ T cells into fully functional effectors. In contrast to bone marrow-derived dendritic cell (BMDC) vaccination, CD40-B cell priming does not allow for memory CD8+ T-cell generation but the reason for this deficiency is unknown. Here, we show that compared to BMDCs, murine CD40-B cells induce lower expression of several genes regulated by T-cell receptor signaling, costimulation, and inflammation (signals 1-3) in mouse T cells. The reduced provision of signals 1 and 2 by CD40-B cells can be explained by a reduction in the quality and duration of the interactions with naive CD8+ T cells as compared to BMDCs. Furthermore, CD40-B cells produce less inflammatory mediators, such as IL-12 and type I interferon, and increasing inflammation by coadministration of polyriboinosinic-polyribocytidylic acid with CD40-B-cell immunization allowed for the generation of long-lived and functional CD8+ memory T cells. In conclusion, it is possible to manipulate CD40-B-cell vaccination to promote the formation of long-lived functional CD8+ memory T cells, a key step before translating the use of CD40-B cells for therapeutic vaccination.

VEGF Requires the Receptor NRP-1 To Inhibit Lipopolysaccharide-Dependent Dendritic Cell Maturation.

To stimulate a productive T cell response, dendritic cells (DC) must undergo maturation characterized by heightened cell surface expression of MHC and costimulatory molecules as well as cytokine production. Conversely, the inhibition of DC maturation is a central mechanism of immune tolerance. The control of the DC maturation process relies on the integration of several cellular stimulatory or inhibitory signals. The soluble factors and their receptors controlling this central aspect of DC biology are incompletely characterized. We show that murine bone marrow-derived DC (BMDC) maturation induced by LPS, as opposed to polyinosinic:polycytidylic acid or cytosine-phosphate-guanine, is robustly inhibited by vascular endothelial growth factor (VEGF), a previously identified immunosuppressive cytokine. Using BMDC from wild type and conditional knockout mice, we show that neuropilin-1 (NRP-1), a known receptor of VEGF, is necessary to suppress LPS-dependent BMDC maturation. The absence of NRP-1 had no ostensible effects on the biology of BMDC in the absence of VEGF. However, NRP-1-deficient BMDC remained completely insensitive to the VEGF-dependent inhibition of BMDC maturation in culture. In the presence of VEGF, NRP-1 directly interacted with the LPS receptor TLR4 and suppressed downstream signaling through ERK and NF-κß, resulting in a sharp inhibition of MHC class II and costimulatory molecules (CD40, CD86) expression as well as proinflammatory cytokine production. Consequently, we identify NRP-1 as a target to optimize DC maturation within environments that are rich in VEGF, such as tumors.

Circadian Clocks in the Immune System.

The immune system is a complex set of physiological mechanisms whose general aim is to defend the organism against non-self-bodies, such as pathogens (bacteria, viruses, parasites), as well as cancer cells. Circadian rhythms are endogenous 24-h variations found in virtually all physiological processes. These circadian rhythms are generated by circadian clocks, located in most cell types, including cells of the immune system. This review presents an overview of the clocks in the immune system and of the circadian regulation of the function of immune cells. Most immune cells express circadian clock genes and present a wide array of genes expressed with a 24-h rhythm. This has profound impacts on cellular functions, including a daily rhythm in the synthesis and release of cytokines, chemokines and cytolytic factors, the daily gating of the response occurring through pattern recognition receptors, circadian rhythms of cellular functions such as phagocytosis, migration to inflamed or infected tissue, cytolytic activity, and proliferative response to antigens. Consequently, alterations of circadian rhythms (e.g., clock gene mutation in mice or environmental disruption similar to shift work) lead to disturbed immune responses. We discuss the implications of these data for human health and the areas that future research should aim to address.

IL-2 induction of Blimp-1 is a key in vivo signal for CD8+ short-lived effector T cell differentiation.

During infection or vaccination, only a small proportion of CD8(+) T cells differentiate into memory cells. The mechanisms underlying the differentiation of CD8(+) T cells into short-lived effector cells (SLECs) or memory precursor effector cells are poorly defined. It was recently shown in infectious models that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) enhances the formation of SLECs. The factors controlling Blimp-1 expression leading to the in vivo formation of SLECs are still not known. However, it has been shown that cytokines such as IL-2 induce Blimp-1 expression in vitro. In this study, we took advantage of the low-inflammation model of dendritic cell immunization to study the role of the IL-2/Blimp-1 axis in SLEC differentiation as well as the importance of Blimp-1 expression in memory precursor effector cells for proper CD8(+) memory generation. Our results show that Blimp-1 deficiency affects effector differentiation and function in the absence of inflammation. Unexpectedly, memory generation was not affected in Blimp-1-deficient OT-I cells responding to vaccination. In addition, modulation of the bioavailability of IL-2 by injection either of a blocking Ab or of the cytokine, demonstrates a link between IL-2, Blimp-1 induction, and SLEC formation in wild-type cells. Conversely, injection of IL-2 had less effect on Blimp-1-deficient CD8(+) T cells, indicating that the effect of IL-2 on in vivo SLEC differentiation is mediated by Blimp-1. In conclusion, IL-2 induction of Blimp-1 expression is a key regulator of SLEC differentiation in vivo.

The catalytic activity of the mitogen-activated protein kinase extracellular signal-regulated kinase 3 is required to sustain CD4+ CD8+ thymocyte survival.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4(+) CD8(+) (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3(-/-) thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.

Nanoparticle adjuvant sensing by TLR7 enhances CD8+ T cell-mediated protection from Listeria monocytogenes infection.

Developing new adjuvants and vaccination strategies is of paramount importance to successfully fight against many life-threatening infectious diseases and cancer. Very few adjuvants are currently authorized for human use, and these mainly stimulate a humoral response. However, specific Abs are not sufficient to confer protection against persisting infections or cancer. Therefore, development of adjuvants and immunomodulators able to enhance cell-mediated immune responses represents a major medical need. We recently showed that papaya mosaic virus nanoparticles (PapMV), self-assembled from the coat protein of a plant virus and a noncoding ssRNA molecule, are highly immunogenic in mice. PapMV can be used either as a vaccine delivery platform, through fusion of various epitopes to the coat protein or as adjuvant to enhance humoral immune responses against coadministered Ags or vaccines. However, the mechanisms that confer these immunomodulatory properties to PapMV and its ability to enhance T cell vaccines remain unknown. Using immunization studies in mice, we demonstrate in this paper that PapMV represents a novel TLR7 agonist with strong immunostimulatory properties. More importantly, pretreatment with PapMV significantly improves effector and memory CD8(+) T cell responses generated through dendritic cell vaccination increasing protection against a Listeria monocytogenes challenge.