Day/night variations of myeloid and lymphoid cell subsets in the murine inguinal lymph node.

Hepatitis B is a noncytopathic virus, which exclusively replicates within the liver and affects 350 million people worldwide [1]. Chronic infection can lead to variable disease manifestations such as cirrhosis, decompensated liver disease and hepatocellular carcinoma causing 1 million deaths per year [2]. These important clinical outcomes are a consequence of the host immune response to HBV, which constitutes a double-edged sword responsible for both viral clearance and hepatocellular damage. The precipitants for natural history milestones such as HBV-related hepatic flares (HF) and hepatitis B e Antigen (HBeAg) seroconversion remain unknown. Virus-specific and non-specific cytotoxic T lymphocytes (CTLs), T regulatory (Treg) cells, Natural Killer (NK), Natural Killer T (NKT) cells and dendritic cells (DCs) have been postulated to play a role [3]. The contribution of these immune cells and the nature of their interaction in the immune pathogenesis of HBV-related liver disease require further characterization. The logistical restraints of longitudinal, peripheral and intrahepatic sampling of the human host as well as inadequate small animal and cell culture models have hampered investigation of these immune mechanisms. As a caveat to human based studies in HBV, the circulating immunological cells may not reflect the phenotype and function of equivalent cells sequestered within the human liver. Current knowledge about the immune response to HBV is extrapolated from transgenic mouse models, many of which are models of viral replication rather than liver injury. Natural Killer (NK) and Natural Killer T (NKT) cells are cytotoxic lymphocytes that constitute a key effector arm of the innate immune system. Efforts to characterise the immunological determinants of Hepatitis B virus (HBV) infection have focused on the adaptive immune system whilst overlooking the potential interaction between virus, hepatocyte and NK or NKT cells, which play an important role in host defense against viral pathogens through direct cytotoxicity and the production of proinflammatory and immune regulatory cytokines. There is debate as to whether NK or NKT cells are effectors of antiviral activity or mediators of hepatic injury and fibrogenesis in chronic hepatitis B infection. NK and NKT cells have been implicated in the pathogenesis of liver disease due to other hepatotropic viruses such as hepatitis C and E as well as autoimmune liver disease, as shown in animal models of liver injury [4]. Human intrahepatic lymphocytes consist of 30%-50% NK and 5-10% NKT cells. Peripheral blood lymphocytes contain 13% NK cells and 2% NKT cells [5]. The hepatic enrichment of NK and NKT cells reflects their role as regulators at the interface between the innate and adaptive immune response to liver disease. NK and NKT cells in the peripheral and intrahepatic compartments share effector functions such as direct killing of viral-infected cells and cytokine production. The latter is considered the more important effector function in CHB [4]. NK and NKT cells demonstrate reciprocal interactions (“crosstalk”) with hepatic macrophages, Kupffer cells (KC), DCs and T cells as part of an amalgamated immune response to HBV [4]. The role of NK and NKT cells in the initiation and orchestration of a dynamic host immune response against HBV-related liver disease is investigated in this thesis. This hepatotrophic virus has evolved direct and indirect strategies to evade or inhibit the large hepatic reservoir of NK and NKT cells. In this thesis, I will focus on the dynamic phenotype and function of NK and NKT cells throughout the different phases of HBV infection, which so far have been poorly characterized. I will also examine the effect of activated NK and NKT cells on liver injury, fibrosis, and their attenuation following HBV treatment, which remains controversial. Understanding the role of NK and NKT cells in the pathogenesis of CHB may help to develop new biomarkers for disease and treatment activity and design novel immunotherapies.

In adult rat brains, brain-derived neurotrophic factor (BDNF) rhythmically oscillates according to the light-dark cycle and exhibits unique functions in particular brain regions. However, little is known of this subject in juvenile rats. Here, we examined diurnal variation in BDNF and neurotrophin-3 (NT-3) levels in 14-day-old rats. BDNF levels were high in the dark phase and low in the light phase in a majority of brain regions. In contrast, NT-3 levels demonstrated an inverse phase relationship that was limited to the cerebral neocortex, including the visual cortex, and was most prominent on postnatal day 14. An 8-h phase advance of the light-dark cycle and sleep deprivation induced an increase in BDNF levels and a decrease in NT-3 levels in the neocortex, and the former treatment reduced synaptophysin expression and the numbers of synaptophysin-positive presynaptic terminals in cortical layer IV and caused abnormal BDNF and NT-3 rhythms 1 week after treatment. A similar reduction of synaptophysin expression was observed in the cortices of Bdnf gene-deficient mice and Ca(2+)-dependent activator protein for secretion 2 gene-deficient mice with abnormal free-running rhythm and autistic-like phenotypes. In the latter mice, no diurnal variation in BDNF levels was observed. These results indicate that regular rhythms of BDNF and NT-3 are essential for correct cortical network formation in juvenile rodents.

Natural Killer T (NKT) cells play an important role in the immune system as demonstrated by their involvement in tumour surveillance, infection and inflammation. Unlike conventional T cells that recognises peptide antigens when presented by the Major Histocompatibility Complex (pMHC), NKT cells recognise glycolipids, presented by a MHC class I-like molecule (CD1d-α-GalCer) to invoke an immune response. Human semi-invariant NKT cells are unique as the majority of them express a T cell receptor (TCR) bearing an invariant α-chain and restricted β-chain repertoire (Vα24-Jα18;Vβ 11). The mouse orthologue also expresses an invariant α-chain (Vα14-Jα18) but a slightly more diverse β-chain repertoire (Vβ2, Vβ7 and Vβ8.2), where Vβ8.2 is most commonly expressed. The crystal structures of the human NKT TCR-CD1d-α-GalCer and mouse orthologue complexes have provided structural insights into how the NKT TCR can recognise a lipid antigen. While it is clear that the NKT TCR docks CD1d-α-GalCer in a different conformation compared to the TCR-pMHC complexes, there was no clear information on the energetic footprint of the NKT TCR's recognition of CD1d-α-GalCer and also how the NKT TCR can differentiate between closely related α-GalCer analogues to induce a biased cytokine response. In this study, an alanine scanning mutagenesis experiment carried out on the human NKT TCR (Vα24-Jα18;Vβ11) and CD1d, as well as the use of α-GalCer analogues, demonstrated that the Jα18-encoded CDR3α loop and Vβ11-encoded CDR2β loop of the NKT TCR play a crucial role in maintaining its interactions with CD1d-α-GalCer. The minimal usage of only six residues, which are also evolutionary conserved in the mouse NKT TCR (Vα14-Jα18;Vβ8.2), explains the semi-invariant nature of the NKT TCR as well as the basis of NKT cell cross-species reactivity. Furthermore, the interactions these residues made are localised directly above the F′ pocket of CD1d, distal from the galactosyl head group of α-GalCer. The use of α-GalCer analogues that contains glycosyl head group modifications, further demonstrated the lesser energetic contribution the NKT TCR CDR1α loop plays in its interactions with the sugar head group of α-GalCer. In contrast, the NKT TCR CDR3α loop, which makes interactions with α-GalCer as well as CD1d, was determined to be the key CDR loop that is energetically important in glycolipid recognition. NKT cells can differentiate between α-GalCer analogues with small modifications on their glycosyl head group to stimulate a biased T helper (Th) cytokine response. Therefore, the ability of how the mouse Vβ8.2 NKT TCR is able to distinguish between different α-GalCer analogues was further investigated through a combination of biophysical, structural and functional experiments. These data had provided further insight into how NKT cells can recognise and differentiate between structurally similar variants of α-GalCer, also referred to as Altered Glycolipid Ligands (AGLs). The crystal structures of all five NKT TCR-CD1d-AGL complexes revealed minimal structural differences. Variations in terms of affinity and kinetics of the NKT TCR engagement onto CD1d-AGLs as well as differences in cellular responses between AGLs were observed. Modifications on the glycosyl head group of the AGLs, directly impacted NKT cell activation as well as the affinity and t1/2 of the NKT TCR recognition. Furthermore, for these glycosyl head group modified AGLs, ligand potency, as determined by the amount of cytokines produced by the NKT cells, was directly affected by the t1/2 of the NKT TCR-CD1d-AGL interaction. In addition, modifications on the acyl chain of the AGLs do not affect the NKT TCR interaction but reduced NKT cell proliferation. This indicated an alternative antigen processing and presentation pathway for these AGLs by CD1d. On the other hand, truncation of the sphingosine chain resulted in a reduction of NKT TCR affinity resulting in an induced-fit mechanism by the NKT TCR. Collectively, the minimal binding requirements of CD1d restriction as well as the molecular basis of NKT fine specificity in CD1d-AGLs recognition were elucidated.

β-Glycoglycosphingolipid-Induced Alterations of the STAT Signaling Pathways Are Dependent on CD1d and the Lipid Raft Protein Flotillin-2

Natural killer T cells are important in the pathogenesis of asthma: The many pathways to asthma

Research Highlights: Immunotherapy

Innate immunity, purinergic system, and liver regeneration: A trip in complexity

Labor resembles an inflammatory response that includes secretion ofcytokines/chemokines by resident and infiltrating immune cells into reproductivetissues and the maternal/fetal interface. Untimely activation of these inflammatorypathways leads to preterm labor, which can result in preterm birth. Preterm birth isa major determinant of neonatal mortality and morbidity; therefore, the elucidationof the process of labor at a cellular and molecular level is essential forunderstanding the pathophysiology of preterm labor. Here, we summarize the role ofinnate and adaptive immune cells in the physiological or pathological activation oflabor. We review published literature regarding the role of innate and adaptiveimmune cells in the cervix, myometrium, fetal membranes, decidua and the fetus inlate pregnancy and labor at term and preterm. Accumulating evidence suggests thatinnate immune cells (neutrophils, macrophages and mast cells) mediate the process oflabor by releasing pro-inflammatory factors such as cytokines, chemokines and matrixmetalloproteinases. Adaptive immune cells (T-cell subsets and B cells) participate inthe maintenance of fetomaternal tolerance during pregnancy, and an alteration intheir function or abundance may lead to labor at term or preterm. Also, immune cellsthat bridge the innate and adaptive immune systems (natural killer T (NKT) cells anddendritic cells (DCs)) seem to participate in the pathophysiology of preterm labor.In conclusion, a balance between innate and adaptive immune cells is required inorder to sustain pregnancy; an alteration of this balance will lead to labor at termor preterm.

The immune system plays a major role in the elimination of tumors. CD8 T cell infiltration is known to be a good prognostic indicator. The development of therapies based on checkpoint inhibitor antibodies was an important breakthrough in increasing survival by limiting the exhaustion of cytotoxic cells and increasing tumor cell eradication. However, the immune system also contains regulatory cells that protect the organism from inappropriate activation of immune cells against self-antigens. Thus, the fact that tumor cells are autologous leads regulatory cells to inhibit the activation of anti-tumor cytotoxic cells and thus increase tumor escape. Therefore, understanding the function and the activation of regulatory cells might help to develop therapies to limit the activation of regulatory cells in order to increase tumor clearance. Natural killer T (NKT) cells are lymphocytes with features of natural killer (NK) and of T cells placing them at the interface of innate and adaptive immunity. Like NK cells, they rapidly produce cytokines after stimulation, orienting the immune response. As T cells, they express a T cell receptor (TCR) that allows the recognition of specific lipids presented by the non-classical MHC-I molecule CD1d. According to their TCR usage, two populations of NKT cells are described: type I and type II. All type I NKT cells express a semi-invariant TCR (Valpha24Jalpha18 in humans, Valpha14Jalpah18 in mice) that recognizes α-galactosylceramide (αGalCer). They can be identified with the αGalCer-loaded CD1d tetramer. In contrast, type II NKT cells express a more diverse TCR repertoire. There is no currently identified lipid antigen recognized by all type II NKT cells, making their identification more difficult. A fraction of them recognize sulfatide. By using sulfatide-loaded CD1d-tetramers, we observed for the first time that sulfatide-reactive type II NKT cells were enriched in the lung, a major site of tumor metastasis. Moreover, we previously showed that in vivo stimulation of type II NKT cells with sulfatide increased the number of tumor nodules in the lung. An in-depth phenotype analysis revealed that they were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells are either CD4+CD8- or double negative. Type II NKT cells do not express PLZF, the master regulator of NKT cell development and exist in PLZF-/- mice contrary to type I NKT cells. We showed that type II NKT cells also expressed markers of myeloid cells, c-Kit, CD11b and Ly6C even though histological analysis revealed lymphocytic morphology. Interestingly, at steady state, type II NKT cells expressed granzyme A but not granzyme B or perforin. The in vivo injection of sulfatide increased the expression of the activation markers CD69 and CD44 as well as granzyme A. Since the regulatory functions of type II NKT cells have been shown to be critical in tumor immunity, the detailed characterization of these cells could help to develop a new immunotherapy for cancer. Citation Format: Lise Pasquet, Shingo Kato, Tony Adams, Susan Sharrow, Theresa Davies-Hill, Elaine Jaffe, Xia Zheng, Motoshi Suzuki, Damian Kovalovsky, Jay Berzofsky, Masaki Terabe. Characterization of sulfatide reactive type II NKT cells from mouse lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2017-4622

Immune privilege in the eye is, in part, associated with the development of an antigen-specific systemic tolerance termed anterior chamber-associated immune deviation (ACAID). Natural killer T (NKT) cells express T-cell receptor and natural killer (NK) markers and are classified as innate immune cells partly because they produce cytokines within minutes of signals. The aim of this study was to elucidate the role of murine NKT cells in the induction of T regulatory cells in anterior chamber-associated immune deviation. Anterior chamber-associated immune deviation T regulatory cell generation ability was examined in the following NKT cell-deficient mice: SJL mice, CDld or Jalpha281 knockout (KO) mice on C57BL/6 (B6) background, and NKT cell-depleted mice. To detect T regulatory cells, splenic T cells were harvested 7 days after anterior chamber inoculation of ovalbumin (50 microg/2 microL in Hanks balanced salt solution [HBSS]), mixed with ovalbumin-primed T cells (effector) and ovalbumin-pulsed antigen-presenting cells (stimulator), and then cotransferred into the ear pinnae of a syngeneic naive mouse (local adoptive transfer assay). Ear swelling was measured 24 hours later. Anterior chamber-inoculated B6 mice developed T regulatory cells, but all natural killer T cell-deficient mice did not generate T regulatory cells unless they were reconstituted with natural killer T cells. We also found that the number of splenic natural killer T cells were increased in anterior chamber-inoculated B6 mice and those natural killer T cells produced IL-10. CD1d-reactive natural killer T cells are essential for the induction of T regulatory cells in anterior chamber-associated immune deviation through their IL-10 production and are involved in the maintenance of immune privilege of the eye.

Starting Over: The Search for Endogenous NKT Cell Ligands

Natural killer T (NKT) cells are specialized CD1d-restricted T cells that recognize lipid antigens. Following stimulation, NKT cells lead to downstream activation of both innate and adaptive immune cells in the tumor microenvironment. This has impelled the development of NKT cell-targeted immunotherapies for treating cancer. In this review, we provide a brief overview of the stimulatory and regulatory functions of NKT cells in tumor immunity as well as highlight preclinical and clinical studies based on NKT cells. Finally, we discuss future perspectives to better harness the potential of NKT cells for cancer therapy.

Pancreatic cancer (PC) is seldom detected at early stages and most PC tumors are unresectable. The five-year survival rate for PC is <6%. Natural Killer (NK) and Natural Killer T (NKT) cell dysfunction is linked to aggressive tumor growth and poor PC prognosis. NKT cells are known to regulate pro-inflammatory responses of tumor-associated macrophages (TAM). We have shown that pancreatic tumors are high in TAM and overexpress microsomal prostaglandin synthase-1 (mPGES-1). Thus, we performed experiments to clarify the roles of NK, NKT, TAM, and mPGES-1 in K-rasG12D driven pancreatic tumor growth in mice. To understand the regulatory role of NKs, we initially crossed Rag1 mice, which have NK cells and lack T cells, with Kras (p48Cre/+-LSL-KrasG12D/+) mice to generate Rag1-Kras mice. At seven months of age, the Rag1-Kras mice showed reduced pancreatic intraepithelial neoplasia (PanIN) lesions without evidence of carcinoma compared with Kras mice (p<0.0001) which displayed high PanINs (p<0.001) and invasive carcinoma. Flow cytometry (FC) analysis of pancreatic tumors (PTs) showed 75% NK cells (NKp46and NK1.1-positive) in PTs of Rag1-Kras mice compared with 6.7% NK cells in PTs from K-ras mice. These results suggest an inhibitory role of NKs on PT. To further study the role of NKTs, we crossbred CD1d−/− mice with K-ras mice, and generated CD1d−/−-Kras mice deficient in both iNKT and vNKT cells. At 5 months of age, the PTs were analyzed histologically, PTs and spleens were analyzed by FC for macrophages (CD68, stabilin) and mPGES-1 expressions, along with stem-like cells (Epcam-, Dclk1-, and Lgr5- positive cells). CD1d−/−-Kras mice were observed to have significantly increased PT weights, and a 50% increase in total PanINs compared with Kras mice (PanIN1, 175 Vs 362, p<0.0001; PanIN2, 80 Vs 162, p<0.0003; PanIN 3, 17 vs 30, p<0.008). PTs and spleens from CD1d−/−-Kras mice showed significantly increased mPGES-1 expression in M2 type macrophages (p<0.005) compared with Kras mice. Tumors from CD1d−/−-Kras mice had more cancer stem-like cells that were positive for Epcam, Dclk-1, and Lgr5 (25%, p<0.02) compared with tumors from Kras mice. To further confirm the relationship between NKT cells, M2-derived mPGES-1, and PC development, we treated CD1d−/−-Kras mice with mPGES-1-specific inhibitor YS-121 after PanIN lesions formed at 12 weeks of age. Mice were killed after 22 weeks of age. We analyzed PanIN formation and PC. Inhibition of mPGES-1 led to a decrease in PanINs and PC development. Thus, the absence or functional loss of NKT cells leads to increased TAMs (M2) with high mPGES-1, resulting in enhanced pancreatic tumor growth and invasion. These results suggest that NKT cells play a regulatory role on macrophages during Kras-induced pancreatic tumor progression. {Supported in part by Kerley-Cade Chair Endowment and NCI-CN-53300}. Citation Format: Naveena B. Janakiram, Altaf Mohammed, Taylor Bryant, Rebekah Ritchie, Gopal Pathuri, Stan Lightfoot, Mark L. Lang, Chinthalapally V. Rao. Enhanced pancreatic tumor progression in p48Cre-KrasG12D mice is regulated by natural killer T (NKT) cells dependent on mPGES-1 in tumor-associated macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3179. doi:10.1158/1538-7445.AM2015-3179

Background: CTCs play a crucial role in tumor dissemination and are prognostic factor in primary and metastatic breast cancer patients. Immune cells in peripheral blood (PB) contribute to an unfavorable microenvironment for the CTCs survival. As such, effective host innate and adaptive immune surveillance systems could adversely influence tumor dissemination whereas dysfunctional immune systems could provide a favorable microenvironment for the dissemination of CTCs and cancer progression. This study aimed to correlate CTCs with the functions of innate [natural killer (NK) cells] and adaptive (T-cells) immune effector cells in PB of IBC patients. Methods: This prospective study included 65 IBC (21 non-metastatic, 14 de novo metastatic and 30 recurrent metastatic) patients treated between October 2008 and April 2012 at the MD Anderson Cancer Center. CTCs were enumerated before patients started a new line of chemotherapy using the CellSearch® system, 33 (50.8%) of patients were treatment naïve at the time of blood collection. The phenotype of T cells, their ability to secrete cytokines following activation through the T-cell receptor (TCR) and the NK cell subsets were analyzed by multiparameter flow cytometry and the results were correlated with CTCs and clinical outcome. For survival analysis immune cell counts were dichotomized to low or high category using the median count. Results: At least 1 CTC (≥ 1) or ≥ 5 CTCs per 7.5 mL of PB was detected in 40 (61.5%) or 21 (32.3%) of patients, respectively. Patients with at least 1 CTC or ≥ 5 CTCs had a significantly inferior overall survival (OS) [HR=2.48, p=0.003 and HR=1.85, p=0.045] than patients with no CTCs or with <5 CTCs, respectively. There was no correlation between CTCs count and total lymphocytes; however, patients with at least 1 CTC or ≥5 CTCs had significantly lower percentages of CD3+ and CD4+ T-cells compared with patients with no CTCs or < 5 CTCs, respectively. Patients with ≥ 1 CTC, had a lower percentage of TCR-activated CD8+ T-cells producing TNF-α (p=0.03) and IFN-γ (p=0.08), and a higher percentage of T regulatory lymphocytes (p=0.05) compared to patients with undetectable CTCs. Moreover, CTCs ≥5 was inversely associated with the percentage of the following NK cells subsets: non-ADCC NK (Spearman rho' = -0.30, p=0.02), ADCC NK (rho' = -0.15, p=0.20) and exhausted NK (rho = -0.24, p=0.04). We also observed increased prognostic value of CTCs in the context of adaptive immune cells, with worse OS for patients with ≥ 5 CTCs and low count of TCR-activated CD8+ T cells producing TNF-α (HR=6.72, p=0.0007) compared with patients with < 5 CTCs and high count of TCR-activated CD8+ that produced TNF-α. Conclusions: IBC patients with CTCs in PB had abnormalities in both innate and adaptive immunity as evidenced by low percentages of NK cell subsets, and low percentage of TCR-activated CD8+ T cells producing TNF-α, respectively. These data illustrate an inverse relationship between CTCs and both innate and adaptive immune cells in the PB microenvironment that could potentially impact tumor cell dissemination and initiation of the metastatic cascade. Moreover, immune cell profiling could add further prognostic value to CTCs in IBC patients. Citation Format: Michal Mego, Hui Gao, Evan N Cohen, Simone Anfossi, Antonio Giordano, Sanda Tin, Tamer M Fouad, Wendy A Woodward, Ricardo H Alvarez, Vicente Valero, Naoto T Ueno, Gabriel N Hortobagyi, Massimo Cristofanilli, James M Reuben. Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-04.

<div>Abstract<p>The physiologic function of natural killer T (NKT) cells in adaptive immunity remains largely unknown because most studies have used NKT cell agonists. In the present study, the role of NKT cells during the secondary effector phase was investigated separately from the primary immunization phase via adoptive transfer of differentiated effector T cells into naive recipients. We found that secondary antitumor CD8<sup>+</sup> T-cell responses were optimal when NKT cells were present. Tumor-specific CD8<sup>+</sup> effector T cells responded less strongly to tumor cell challenge in NKT cell–deficient recipients than in recipients with intact NKT cells. NKT cell–mediated enhancement of the secondary antitumor CD8<sup>+</sup> T-cell response was concurrent with increased number and activity of tumor-specific CD8<sup>+</sup> T cells. These findings provide the first demonstration of a direct role for NKT cells in the regulation of antigen-specific secondary T-cell responses without the use of exogenous NKT cell agonists such as α-galactosylceramide (α-GalCer). Furthermore, forced activation of NKT cells with α-GalCer during the secondary immune response in suboptimally immunized animals enhanced otherwise poor tumor rejection responses. Taken together, our findings strongly emphasize the importance of NKT cells in secondary CD8<sup>+</sup> T-cell immune responses. [Cancer Res 2009;69(10):4301–8]</p></div>