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.

NR4A3 Mediates Thymic Negative Selection.

Central tolerance aims to limit the production of T lymphocytes bearing TCR with high affinity for self-peptide presented by MHC molecules. The accumulation of thymocytes with such receptors is limited by negative selection or by diversion into alternative differentiation, including T regulatory cell commitment. A role for the orphan nuclear receptor NR4A3 in negative selection has been suggested, but its function in this process has never been investigated. We find that Nr4a3 transcription is upregulated in postselection double-positive thymocytes, particularly those that have received a strong selecting signal and are destined for negative selection. Indeed, we found an accumulation of cells bearing a negative selection phenotype in NR4A3-deficient mice as compared with wild-type controls, suggesting that Nr4a3 transcriptional induction is necessary to limit accumulation of self-reactive thymocytes. This is consistent with a decrease of cleaved caspase-3+-signaled thymocytes and more T regulatory and CD4+Foxp3-HELIOS+ cells in the NR4A3-deficient thymus. We further tested the role for NR4A3 in negative selection by reconstituting transgenic mice expressing the OVA Ag under the control of the insulin promoter with bone marrow cells from OT-I Nr4a3 +/+ or OT-I Nr4a3 -/- mice. Accumulation of autoreactive CD8 thymocytes and autoimmune diabetes developed only in the absence of NR4A3. Overall, our results demonstrate an important role for NR4A3 in T cell development.

Early programming of CD8+ T cell response by the orphan nuclear receptor NR4A3.

Enhancing long-term persistence while simultaneously potentiating the effector response of CD8+ T cells has been a long-standing goal in immunology to produce better vaccines and adoptive cell therapy products. NR4A3 is a transcription factor of the orphan nuclear receptor family. While it is rapidly and transiently expressed following T cell activation, its role in the early stages of T cell response is unknown. We show that NR4A3-deficient murine CD8+ T cells differentiate preferentially into memory precursor and central memory cells, but also produce more cytokines. This is explained by an early influence of NR4A3 deficiency on the memory transcriptional program and on accessibility of chromatin regions with motifs for bZIP transcription factors, which impacts the transcription of Fos/Jun target genes. Our results reveal a unique and early role for NR4A3 in programming CD8+ T cell differentiation and function. Manipulating NR4A3 activity may represent a promising strategy to improve vaccination and T cell therapy.

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.

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.

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.

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.

The atypical MAPK ERK3 controls positive selection of thymocytes.

Extracellular signal-regulated kinase 3 (ERK3 )is an atypical member of the mitogen-activated protein kinase (MAPK) family. We have previously shown that ERK3 is expressed during thymocyte differentiation and that its expression is induced in mature peripheral T cells following activation of ERK1/2 by T-cell receptor (TCR) signalling. Herein, we have investigated whether ERK3 expression is required for proper T-cell selection. Using a knock-in mouse model in which the coding sequence of ERK3 is replaced by the gene encoding for the ß-galactosidase reporter, we show that ERK3 is expressed by double-positive (DP) thymocytes undergoing positive selection. In ERK3-deficient mice with a polyclonal TCR repertoire, we observe a decrease in positive selection. This reduction in positive selection was also observed when ERK3-deficient mice were backcrossed to class I- and class II-restricted TCR transgenic mice. Furthermore, the response of DP thymocytes to in vitro TCR stimulation was strongly reduced in ERK3-deficient mice. Together, these results show that ERK3 expression following TCR signalling is critical for proper thymic positive selection.

Neuropilin-1 mediates myeloid cell chemoattraction and influences retinal neuroimmune crosstalk.

Immunological activity in the CNS is largely dependent on an innate immune response and is heightened in diseases, such as diabetic retinopathy, multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease. The molecular dynamics governing immune cell recruitment to sites of injury and disease in the CNS during sterile inflammation remain poorly defined. Here, we identified a subset of mononuclear phagocytes (MPs) that responds to local chemotactic cues that are conserved among central neurons, vessels, and immune cells. Patients suffering from late-stage proliferative diabetic retinopathy (PDR) had elevated vitreous semaphorin 3A (SEMA3A). Using a murine model, we found that SEMA3A acts as a potent attractant for neuropilin-1-positive (NRP-1-positive) MPs. These proangiogenic MPs were selectively recruited to sites of pathological neovascularization in response to locally produced SEMA3A as well as VEGF. NRP-1-positive MPs were essential for disease progression, as NRP-1-deficient MPs failed to enter the retina in a murine model of oxygen-induced retinopathy (OIR), a proxy for PDR. OIR mice with NRP-1-deficient MPs exhibited decreased vascular degeneration and diminished pathological preretinal neovascularization. Intravitreal administration of a NRP-1-derived trap effectively mimicked the therapeutic benefits observed in mice lacking NRP-1-expressing MPs. Our findings indicate that NRP-1 is an obligate receptor for MP chemotaxis, bridging neural ischemia to an innate immune response in neovascular retinal disease.

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.

The non-classical MAP kinase ERK3 controls T cell activation.

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

IL-6 production by dendritic cells is dispensable for CD8+ memory T-cell generation.

Following activation, naïve CD8(+) T cells will differentiate into effectors that differ in their ability to survive: some will persist as memory cells while the majority will die by apoptosis. Signals given by antigen-presenting cells (APCs) at the time of priming modulate this differential outcome. We have recently shown that, in opposition to dendritic cell (DC), CD40-activated B-(CD40-B) cell vaccination fails to efficiently produce CD8(+) memory T cells. Understanding why CD40-B-cell vaccination does not lead to the generation of functional long-lived memory cells is essential to define the signals that should be provided to naïve T cells by APCs. Here we show that CD40-B cells produce very low amount of IL-6 when compared to DCs. However, supplementation with IL-6 during CD40-B-cell vaccination did not improve memory generation. Furthermore, IL-6-deficient DCs maintained the capacity to promote the formation of functional CD8(+) effectors and memory cells. Our results suggest that in APC vaccination models, IL-6 provided by the APCs is dispensable for proper CD8(+) T-cell memory generation.

Inhibitory Killer Immunoglobulin-like Receptors to self HLA-B and HLA-C ligands contribute differentially to Natural Killer cell functional potential in HIV infected slow progressors.

Inhibitory Killer Immunoglobulin-like Receptors (iKIR) interact with their ligands, HLA molecules, to license Natural Killer (NK) cells for functional competence. Previous studies stimulating peripheral blood mononuclear cells (PBMCs) with the HLA-devoid K562 cell line revealed that NK cells from individuals with an iKIR encoded by the KIR3DL1 locus with self HLA-Bw4 as their ligands, had higher frequencies of tri-functional NK cells that expressed the degranulation marker CD107a and secreted Interferon-γ and Tumor Necrosis Factor-α than those from individuals who were homozygous for HLA-Bw6 alleles, which are not ligands for these iKIR. To assess the effect of other iKIR to self-HLA (S-iKIR) on the NK cell response, we compared HIV-infected slow progressors (SP) carrying S-iKIR to HLA-C alleles with or without S-iKIR to HLA-Bw4. We show that S-iKIR to HLA-B and C alleles differ in their contribution to NK cell functional potential in HIV-infected SP upon stimulation with K562 targets.

CD40-activated B cells can efficiently prime antigen-specific naïve CD8+ T cells to generate effector but not memory T cells.

BACKGROUND: The identification of the signals that should be provided by antigen-presenting cells (APCs) to induce a CD8(+) T cell response in vivo is essential to improve vaccination strategies using antigen-loaded APCs. Although dendritic cells have been extensively studied, the ability of other APC types, such as B cells, to induce a CD8(+) T cell response have not been thoroughly evaluated. METHODOLOGY/PRINCIPAL FINDINGS: In this manuscript, we have characterized the ability of CD40-activated B cells, stimulated or not with Toll-like receptor (TLR) agonists (CpG or lipopolysaccharide) to induce the response of mouse naïve CD8(+) T cells in vivo. Our results show that CD40-activated B cells can directly present antigen to naïve CD8(+) T cells to induce the generation of potent effectors able to secrete cytokines, kill target cells and control a Listeria monocytogenes infection. However, CD40-activated B cell immunization did not lead to the proper formation of CD8(+) memory T cells and further maturation of CD40-activated B cells with TLR agonists did not promote the development of CD8(+) memory T cells. Our results also suggest that inefficient generation of CD8(+) memory T cells with CD40-activated B cell immunization is a consequence of reduced Bcl-6 expression by effectors and enhanced contraction of the CD8(+) T cell response. CONCLUSIONS: Understanding why CD40-activated B cell immunization is defective for the generation of memory T cells and gaining new insights about signals that should be provided by APCs are key steps before translating the use of CD40-B cell for therapeutic vaccination.

T cell Activation does not drive CD4 decline in longitudinally followed HIV-infected Elite Controllers.

BACKGROUND: Elite controllers (EC) are a rare subset of HIV infected individuals who control viral load below 50 copies/ml of plasma without treatment. METHODS: Thirty four EC were studied. The slope of CD4 count change was available for 25 of these subjects. We assessed immune activation by measuring the percent of CD38+HLA-DR+CD8+ T cells in the EC group and comparing it with that in 24 treatment-naïve HIV disease progressors and 13 HIV uninfected healthy controls. RESULTS: Compared to HIV uninfected subjects, EC had higher percentages of CD38+HLA-DR+CD8+ T cells (p < 0.001) that was lower than that observed in progressors (p < 0.01). Fifteen of 25 EC had a slope of CD4 count change that was not significantly different from 0 while 3 had a positive and 7 a negative CD4 count slope. Immune activation did not distinguish EC subsets with stable/increasing versus declining CD4 counts. CONCLUSIONS: Elevated immune activation in ECs is not associated with a faster rate of CD4 decline.

Receptor-ligand requirements for increased NK cell polyfunctional potential in slow progressors infected with HIV-1 coexpressing KIR3DL1*h/*y and HLA-B*57.

Carriage of the natural killer (NK) receptor genotype KIR3DL1*h/*y with its HLA-B*57 ligand (*h/*y+B*57) is associated with slow time to AIDS and low viral load (VL). To provide a functional basis for these epidemiological observations, we assessed whether HIV-1-infected slow progressors (SP) carrying the *h/*y+B*57 compound genotype would have increased NK cell polyfunctional potential in comparison to SP with other killer immunoglobulin-like receptor (KIR)/HLA compound genotypes and whether this enhanced polyfunctionality was dependent upon the coexpression of both KIR3DL1*h/*y and HLA-B*57. The functional potential of NK cells was investigated by stimulating peripheral blood mononuclear cells with HLA-devoid targets or single HLA transfectants. Multiparametric flow cytometry was used to detect NK cells with seven functional profiles representing all permutations of CD107a expression and gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) secretion. NK cells from individuals carrying KIR3DL1 receptor-HLA-Bw4 ligand pairs had greater trifunctional responses than those from KIR3DL1 homozygotes (hmz), who were Bw6 homozygotes. NK cells from subjects carrying the *h/*y+B*57 genotypes exhibited the highest trifunctional potential, and this was dependent on cocarriage of the NK receptor and its ligand. Trifunctional cells secreted more of each function tested on a per-cell basis than each corresponding monofunctional NK subset. Although VL influenced NK functionality, individuals with defined KIR/HLA genotypes exhibited differences in NK cell polyfunctionality that could not be accounted for by VL alone. The protective effect of HLA-B*57 on slow progression to AIDS and low VL may be mediated through its interaction with KIR3DL1 alleles to educate NK cells for potent activity upon stimulation.

Mind the gap: lack of association between KIR3DL1*004/HLA­Bw4-induced natural killer cell function and protection from HIV infection.

Several combinations of genes encoding KIR3DL1 alleles and their HLA­Bw4 ligands have been linked with favorable outcomes upon exposure to or infection with human immunodeficiency virus (HIV). Some protective KIR3DL1/HLA­Bw4 combinations confer elevated natural killer (NK) cell functional potential. The K562­stimulated functionality of NK cells from KIR3DL1*004/HLA­Bw4 and control genotype carriers was assessed by flow cytometry and found to be higher in KIR3DL1*004/HLA­Bw4 carriers. However, a comparison of the frequency of this combined genotype among HIV­exposed uninfected and HIV­infected subjects revealed no between­group differences. Thus, despite its ability to license NK cells, KIR3DL1*004/HLA­Bw4 is not associated with a reduced risk of infection.