Bacillus anthracis Lethal Toxin Disrupts TCR Signaling in CD1d-Restricted NKT Cells Leading to Functional Anergy

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen

Dendritic cells license regulatory B cells to produce IL-10 and mediate suppression of antigen-specific CD8 T cells

Regulatory B cells (Bregs) suppress and reduce autoimmune pathology. However, given the variety of Breg subsets, the role of Bregs in the pathogenesis of type 1 diabetes is still unclear. Here, we dissect this fundamental mechanism. We show that natural protection from type 1 diabetes in nonobese diabetic (NOD) mice is associated with increased numbers of IL-10-producing B cells, while development of type 1 diabetes in NOD mice occurs in animals with compromised IL-10 production by B cells. However, B cells from diabetic mice regain IL-10 function if activated by the innate immune receptor TLR4 and can suppress insulin-specific CD8 T cells in a dendritic cell (DC)-dependent, IL-10-mediated fashion. Suppression of CD8 T cells is reliant on B-cell contact with DCs. This cell contact results in deactivation of DCs, inducing a tolerogenic state, which in turn can regulate pathogenic CD8 T cells. Our findings emphasize the importance of DC–Breg interactions during the development of type 1 diabetes

B7-H1-Deficiency Enhances the Potential of Tolerogenic Dendritic Cells by Activating CD1d-Restricted Type II NKT Cells

Background: Dendritic cells (DC) can act tolerogenic at a semi-mature stage by induction of protective CD4+ T cell and NKT cell responses. Methodology/Principal Findings: Here we studied the role of the co-inhibitory molecule B7-H1 (PD-L1, CD274) on semimature DC that were generated from bone marrow (BM) cells of B7-H12/2 mice and applied to the model of Experimental Autoimmune Encephalomyelitis (EAE). Injections of B7-H1-deficient DC showed increased EAE protection as compared to wild type (WT)-DC. Injections of B7-H12/2 TNF-DC induced higher release of peptide-specific IL-10 and IL-13 after restimulation in vitro together with elevated serum cytokines IL-4 and IL-13 produced by NKT cells, and reduced IL-17 and IFN-c production in the CNS. Experiments in CD1d2/2 and Ja2812/2 mice as well as with type I and II NKT cell lines indicated that only type II NKT cells but not type I NKT cells (invariant NKT cells) could be stimulated by an endogenous CD1d-ligand on DC and were responsible for the increased serum cytokine production in the absence of B7-H1. Conclusions/Significance: Together, our data indicate that BM-DC express an endogenous CD1d ligand and B7-H1 to ihibit type II but not type I NKT cells. In the absence of B7-H1 on these DC their tolerogenic potential to stimulate tolerogenic CD4+ and NKT cell responses is enhanced

Spleen-Resident CD4+ and CD4− CD8α− Dendritic Cell Subsets Differ in Their Ability to Prime Invariant Natural Killer T Lymphocytes

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α+ and CD8α− cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4− and CD4+ CD8α− cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4− and CD4+ cDC are equivalent in their capacity to prime and direct CD4+ and CD8+ T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4− and CD4+ cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4+ counterparts, CD4− cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4+ and CD4− cDC subsets that may be important in immune responses

Activated iNKT Cells Promote Memory CD8+ T Cell Differentiation during Viral Infection

α-galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8+ T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8+ T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8+ T cells, as a consequence of reduced inflammation

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 cel

Human Natural Killer T Cells Are Heterogeneous in Their Capacity to Reprogram Their Effector Functions

BACKGROUND: Natural killer T (NKT) cells are a subset of T cells that help potentiate and regulate immune responses. Although human NKT cell subsets with distinct effector functions have been identified, it is unclear whether the effector functions of these subsets are imprinted during development or can be selectively reprogrammed in the periphery. RESULTS: We found that neonatal NKT cells are predominantly CD4+ and express higher levels of CCR7 and CD62L and lower levels of CD94 and CD161 than adult CD4+ or CD4− NKT cell subsets. Accordingly, neonatal NKT cells were more flexible than adult CD4+ NKT cells in their capacity to acquire Th1- or Th2-like functions upon either cytokine-mediated polarization or ectopic expression of the Th1 or Th2 transcription factors T-bet and GATA-3, respectively. Consistent with their more differentiated phenotype, CD4- NKT cells were predominantly resistant to functional reprogramming and displayed higher cytotoxic function. In contrast to conventional T cells, neither the expression of CXCR3 nor the cytotoxic capacity of neonatal NKT cells could be reprogrammed. CONCLUSIONS AND SIGNIFICANCE: Together, these results suggest that neonatal CD4+, adult CD4+, and adult CD4− NKT may represent unique states of maturation and that some functions of human NKT cells may be developmentally imprinted, while others are acquired similar to conventional T cell subsets during peripheral maturation and differentiation. Given the potent immuno-regulatory functions of NKT cells, these findings have important implications for the development of novel NKT cell-based therapeutics and vaccines

Abnormal Changes in NKT Cells, the IGF-1 Axis, and Liver Pathology in an Animal Model of ALS

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing fatal neurodegenerative disorder characterized by the selective death of motor neurons (MN) in the spinal cord, and is associated with local neuroinflammation. Circulating CD4+ T cells are required for controlling the local detrimental inflammation in neurodegenerative diseases, and for supporting neuronal survival, including that of MN. T-cell deficiency increases neuronal loss, while boosting T cell levels reduces it. Here, we show that in the mutant superoxide dismutase 1 G93A (mSOD1) mouse model of ALS, the levels of natural killer T (NKT) cells increased dramatically, and T-cell distribution was altered both in lymphoid organs and in the spinal cord relative to wild-type mice. The most significant elevation of NKT cells was observed in the liver, concomitant with organ atrophy. Hepatic expression levels of insulin-like growth factor (IGF)-1 decreased, while the expression of IGF binding protein (IGFBP)-1 was augmented by more than 20-fold in mSOD1 mice relative to wild-type animals. Moreover, hepatic lymphocytes of pre-symptomatic mSOD1 mice were found to secrete significantly higher levels of cytokines when stimulated with an NKT ligand, ex-vivo. Immunomodulation of NKT cells using an analogue of α-galactosyl ceramide (α-GalCer), in a specific regimen, diminished the number of these cells in the periphery, and induced recruitment of T cells into the affected spinal cord, leading to a modest but significant prolongation of life span of mSOD1 mice. These results identify NKT cells as potential players in ALS, and the liver as an additional site of major pathology in this disease, thereby emphasizing that ALS is not only a non-cell autonomous, but a non-tissue autonomous disease, as well. Moreover, the results suggest potential new therapeutic targets such as the liver for immunomodulatory intervention for modifying the disease, in addition to MN-based neuroprotection and systemic treatments aimed at reducing oxidative stress

Tailored design of NKT-stimulatory glycolipids for polarization of immune responses

Natural killer T (NKT) cell is a distinct population of T lymphocytes that can rapidly release massive amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipids presented by CD1d. The secreted cytokines can promote cell-mediated immunity to kill tumor cells and intracellular pathogens, or suppress autoreactive immune cells in autoimmune diseases. Thus, NKT cell is an attractive target for developing new therapeutics to manipulate immune system. The best-known glycolipid to activate NKT cells is α-galactosylceramide (α-GalCer), which has been used as a prototype for designing new NKT stimulatory glycolipids. Many analogues have been generated by modification of the galactosyl moiety, the acyl chain or the phytosphingosine chain of α-GalCer. Some of the analogues showed greater abilities than α-GalCer in polarizing immune responses toward Th1 or Th2 dominance. Among them, several analogues containing phenyl groups in the lipid tails were more potent in inducing Th1-skewed cytokines and exhibited greater anticancer efficacy than α-GalCer. Analyses of the correlation between structure and activity of various α-GalCer analogues on the activation of iNKT cell revealed that CD1d–glycolipid complexes interacted with the same population of iNKT cell expressing similar T-cell receptor Vβ as α-GalCer. On the other hand, those phenyl glycolipids with propensity for Th1 dominant responses showed greater binding avidity and stability than α-GalCer for iNKT T-cell receptor when complexed with CD1d. Thus, it is the avidity and stability of the ternary complexes of CD1d-glycolipid-iNKT TCR that dictate the polarity and potency of immune responses. These findings provide a key to the rationale design of immune modulating glycolipids with desirable Th1/Th2 polarity for clinical application. In addition, elucidation of α-GalCer-induced anergy, liver damage and accumulation of myeloid derived suppressor cells has offered explanation for its lacklustre anti-cancer activities in clinical trials. On other hand, the lack of such drawbacks in glycolipid analogues containing phenyl groups in the lipid tails of α-GalCer coupled with the greater binding avidity and stability of CD1d-glycolipid complex for iNKT T-cell receptor, account for their superior anti-cancer efficacy in tumor bearing mice. Further clinical development of these phenyl glycolipids is warranted