CD1d-mediated Recognition of an α-Galactosylceramide by Natural Killer T Cells Is Highly Conserved through Mammalian Evolution

Natural killer (NK) T cells are a lymphocyte subset with a distinct surface phenotype, an invariant T cell receptor (TCR), and reactivity to CD1. Here we show that mouse NK T cells can recognize human CD1d as well as mouse CD1, and human NK T cells also recognize both CD1 homologues. The unprecedented degree of conservation of this T cell recognition system suggests that it is fundamentally important. Mouse or human CD1 molecules can present the glycolipid α-galactosylceramide (α-GalCer) to NK T cells from either species. Human T cells, preselected for invariant Vα24 TCR expression, uniformly recognize α-GalCer presented by either human CD1d or mouse CD1. In addition, culture of human peripheral blood cells with α-GalCer led to the dramatic expansion of NK T cells with an invariant (Vα24⁺) TCR and the release of large amounts of cytokines. Because invariant Vα14⁺ and Vα24⁺ NK T cells have been implicated both in the control of autoimmune disease and the response to tumors, our data suggest that α-GalCer could be a useful agent for modulating human immune responses by activation of the highly conserved NK T cell subset.     

Small intestinal CD103+ dendritic cells display unique functional properties that are conserved between mice and humans

A functionally distinct subset of CD103⁺ dendritic cells (DCs) has recently been identified in murine mesenteric lymph nodes (MLN) that induces enhanced FoxP3⁺ T cell differentiation, retinoic acid receptor signaling, and gut-homing receptor (CCR9 and α4β7) expression in responding T cells. We show that this function is specific to small intestinal lamina propria (SI-LP) and MLN CD103⁺ DCs. CD103⁺ SI-LP DCs appeared to derive from circulating DC precursors that continually seed the SI-LP. BrdU pulse-chase experiments suggested that most CD103⁺ DCs do not derive from a CD103⁻ SI-LP DC intermediate. The majority of CD103⁺ MLN DCs appear to represent a tissue-derived migratory population that plays a central role in presenting orally derived soluble antigen to CD8⁺ and CD4⁺ T cells. In contrast, most CD103⁻ MLN DCs appear to derive from blood precursors, and these cells could proliferate within the MLN and present systemic soluble antigen. Critically, CD103⁺ DCs with similar phenotype and functional properties were present in human MLN, and their selective ability to induce CCR9 was maintained by CD103⁺ MLN DCs isolated from SB Crohn's patients. Thus, small intestinal CD103⁺ DCs represent a potential novel target for regulating human intestinal inflammatory responses.     

A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity

Airway hypersensitive reaction (AHR) is an animal model for asthma, which is caused or enhanced by environmental factors such as allergen exposure. However, the precise mechanisms that drive AHR remain unclear. We identified a novel subset of natural killer T (NKT) cells that expresses the interleukin 17 receptor B (IL-17RB) for IL-25 (also known as IL-17E) and is essential for the induction of AHR. IL-17RB is preferentially expressed on a fraction of CD4⁺ NKT cells but not on other splenic leukocyte populations tested. IL-17RB⁺ CD4⁺ NKT cells produce predominantly IL-13 and Th2 chemokines upon stimulation with IL-25 in vitro. IL-17RB⁺ NKT cells were detected in the lung, and depletion of IL-17RB⁺ NKT cells by IL-17RB–specific monoclonal antibodies or NKT cell–deficient Jα18^−/− mice failed to develop IL-25–dependent AHR. Cell transfer of IL-17RB⁺ but not IL-17RB⁻ NKT cells into Jα18^−/− mice also successfully reconstituted AHR induction. These results strongly suggest that IL-17RB⁺ CD4⁺ NKT cells play a crucial role in the pathogenesis of asthma.     

Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells

If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex-negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid alpha-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic alphaCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance.     

A comparative study on roles of natural killer T cells in two diet-induced non-alcoholic steatohepatitis-related fibrosis in mice

BackgroundImmune responses are important in the progression of non-alcoholic fatty liver disease (NAFLD). Natural killer T (NKT) cells are main components of the innate immune system that modulate immunity. However, the role of NKT cells in NAFLD remains controversial.ObjectiveWe aimed to investigate the role of NKT cells in non-alcoholic steatohepatitis (NASH)-related fibrosis in fast food diet (FFD)- and methionine choline-deficient (MCD) diet-induced mouse models.MethodsHepatic NKT cells were analysed in wild-type (WT) and CD1d-/- mice fed FFD or MCD diets. Hepatic pathology, cytokine profiles and liver fibrosis were evaluated. Furthermore, the effect of chronic administration of α-galactosylceramide (α-GalCer) on liver fibrosis was investigated in both FFD- and MCD-treated mice.ResultsFFD induced a significant depletion of hepatic NKT cells, thus leading to mild to moderate NASH and early-stage fibrosis, while mice fed MCD diets developed severe liver inflammation and progressive fibrosis without a significant change in hepatic NKT cell abundance. FFD induced a similar liver fibrogenic response in CD1d-/- and WT mice, while MCD induced a higher hepatic mRNA expression of Col1α1 and TIMP1 as well as relative fibrosis density in CD1d-/- mice than WT mice (31.8 vs. 16.3, p = .039; 40.0 vs. 22.6, p = .019; 2.24 vs. 1.59, p = .036). Chronic administration of α-GalCer induced a higher hepatic mRNA expression of TIMP1 in MCD-treated mice than controls (36.7 vs. 14.9, p = .005).ConclusionNKT cells have protective roles in NAFLD as the disease progresses. During diet-induced steatosis, mild to moderate NASH and the early stage of fibrosis, hepatic NKT cells are relatively depleted, leading to a proinflammatory status. In severe NASH and the advanced stage of liver fibrosis, NKT cells play a role in inhibiting the NASH-related fibrogenic response. Chronic administration of α-GalCer induces NKT cell anergy and tolerance, which may play a role in promoting the liver fibrogenic response.     

α/β–T Cell Receptor (TCR)+CD4−CD8− (NKT) Thymocytes Prevent Insulin-dependent Diabetes Mellitus in Nonobese Diabetic (NOD)/Lt Mice by the Influence of Interleukin (IL)-4 and/or IL-10

We have previously shown that nonobese diabetic (NOD) mice are selectively deficient in α/β-T cell receptor (TCR)⁺CD4⁻CD8⁻ NKT cells, a defect that may contribute to their susceptibility to the spontaneous development of insulin-dependent diabetes mellitus (IDDM). The role of NKT cells in protection from IDDM in NOD mice was studied by the infusion of thymocyte subsets into young female NOD mice. A single intravenous injection of 10⁶ CD4^−/lowCD8⁻ or CD4⁻CD8⁻ thymocytes from female (BALB/c × NOD)F1 donors protected intact NOD mice from the spontaneous onset of clinical IDDM. Insulitis was still present in some recipient mice, although the cell infiltrates were principally periductal and periislet, rather than the intraislet pattern characteristic of insulitis in unmanipulated NOD mice. Protection was not associated with the induction of “allogenic tolerance” or systemic autoimmunity. Accelerated IDDM occurs after injection of splenocytes from NOD donors into irradiated adult NOD recipients. When α/β-TCR⁺ and α/β-TCR⁻ subsets of CD4⁻CD8⁻ thymocytes were transferred with diabetogenic splenocytes and compared for their ability to prevent the development of IDDM in irradiated adult recipients, only the α/β-TCR⁺ population was protective, confirming that NKT cells were responsible for this activity. The protective effect in the induced model of IDDM was neutralized by anti–IL-4 and anti–IL-10 monoclonal antibodies in vivo, indicating a role for at least one of these cytokines in NKT cell-mediated protection. These results have significant implications for the pathogenesis and potential prevention of IDDM in humans.     

Mature dendritic cells boost functionally superior CD8(+) T-cell in humans without foreign helper epitopes

We have recently shown that a single injection of mature, antigen-pulsed, human dendritic cells (DCs) rapidly elicits CD4⁺ and CD8⁺ T-cell immunity in vivo. The DCs were pulsed with 2 foreign proteins, keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT), as well as an HLA A2.1-restricted influenza matrix peptide (MP). Responses to all 3 antigens peaked at 30–90 days after immunization and declined thereafter. To determine if the foreign helper proteins (TT and KLH) were essential for CD8⁺ T-cell responses to the viral peptide, we reinjected 3 of the HLA-2.1 subjects with mature DCs pulsed with MP alone. All 3 volunteers showed a rapid boost in MP-specific immunity, and freshly sampled blood from 1 contained cytolytic T cells. In all 3 subjects, CD8⁺ T-cell responses to booster DCs were faster and of greater magnitude than the responses to the first DC injection. Importantly, the T cells that proliferated after booster DC treatment secreted interferon-γ upon challenge with much lower doses of viral peptide than those elicited after the first injection, indicating a higher functional avidity for the ligand. These data begin to outline the kinetics of T-cell immunity in response to DCs and demonstrate that booster injections of mature DCs enhance both qualitative and quantitative aspects of CD8⁺ T-cell function in humans.     

Transforming Growth Factor β1, in the Presence of Granulocyte/Macrophage Colony-stimulating Factor and Interleukin 4, Induces Differentiation of Human Peripheral Blood Monocytes into Dendritic Langerhans Cells

Langerhans cells (LCs) are dendritic cells (DCs) that are present in the epidermis, bronchi, and mucosae. Although LCs originate in bone marrow, little is known about their lineage of origin. In this study, we demonstrate that in vitro LCs may originate from monocytes. Adult peripheral blood CD14⁺ monocytes differentiate into LCs (CD1a⁺, E-cadherin⁺, cutaneous lymphocyte-associated antigen⁺, Birbeck granules⁺, Lag⁺) in the presence of granulocyte/macrophage colony-stimulating factor, interleukin 4, and transforming growth factor β1 (TGF-β1). This process occurs with virtually no cell proliferation and is not impaired by 30 Gy irradiation. Selection of monocyte subpopulations is ruled out since monocyte-derived DCs can further differentiate into LCs. Our data suggest that in vivo LC differentiation may be induced peripherally, from a nonproliferating myeloid precursor, i.e., the monocyte, in response to a TGF-β1–rich microenvironment, as found in the skin and epithelia. Therefore, the monocyte may represent a circulating precursor critical to the immune response in vivo.     

Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

Tumors deficient in expression of the transporter associated with antigen processing (TAP) usually fail to induce T-cell-mediated immunity and are resistant to T-cell lysis. However, we have found that introduction of the B7.1 gene into TAP-negative (TAP⁻) or TAP1-transfected (TAP1⁺) murine lung carcinoma CMT.64 cells can augment the capacity of the cells to induce a protective immune response against wild-type tumor cells. Differences in the strength of the protective immune responses were observed between TAP⁻ and TAP1⁺ B7.1 expressing CMT.64 cells depending on the doses of γ-irradiated cell immunization. While mice immunized with either high or low dose of B7.1-expressing TAP1⁺ cells rejected TAP⁻ tumors, only high dose immunization with B7.1-expressing TAP⁻ cells resulted in tumor rejection. The induced protective immunity was T-cell dependent as demonstrated by dramatically reduced antitumor immunity in mice depleted of CD8 or CD4 cells. Augmentation of T-cell mediated immune response against TAP⁻ tumor cells was also observed in a virally infected tumor cell system. When mice were immunized with a high dose of γ-irradiated CMT.64 cells infected with vaccinia viruses carrying B7.1 and/or TAP1 genes, we found that the cells co-expressing B7.1 and TAP1, but not those expressing B7.1 alone, induced protective immunity against CMT.64 cells. In addition, inoculation with live tumor cells transfected with several different gene(s) revealed that only B7.1- and TAP1-coexpressing tumor cells significantly decreased tumorigenicity. These results indicate that B7.1-provoked antitumor immunity against TAP⁻ cancer is facilitated by TAP1-expression, and thus both genes should be considered for cancer therapy in the future.     

Induction of high-affinity IgE receptor on lung dendritic cells during viral infection leads to mucous cell metaplasia

Respiratory viral infections are associated with an increased risk of asthma, but how acute Th1 antiviral immune responses lead to chronic inflammatory Th2 disease remains undefined. We define a novel pathway that links transient viral infection to chronic lung disease with dendritic cell (DC) expression of the high-affinity IgE receptor (FcεRIα). In a mouse model of virus-induced chronic lung disease, in which Sendai virus triggered a switch to persistent mucous cell metaplasia and airway hyperreactivity after clearance of replicating virus, we found that FceRIa^−/− mice no longer developed mucous cell metaplasia. Viral infection induced IgE-independent, type I IFN receptor–dependent expression of FcεRIα on mouse lung DCs. Cross-linking DC FcεRIα resulted in the production of the T cell chemoattractant CCL28. FceRIa^−/− mice had decreased CCL28 and recruitment of IL-13–producing CD4⁺ T cells to the lung after viral infection. Transfer of wild-type DCs to FceRIa^−/− mice restored these events, whereas blockade of CCL28 inhibited mucous cell metaplasia. Therefore, lung DC expression of FcεRIα is part of the antiviral response that recruits CD4⁺ T cells and drives mucous cell metaplasia, thus linking antiviral responses to allergic/asthmatic Th2 responses.     

Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells

NKT cells demonstrate antitumor activity when activated to produce Th1 cytokines by DCs loaded with α-galactosylceramide, the prototypic NKT cell–activating glycolipid antigen. However, most patients do not have sufficient numbers of NKT cells to induce an effective immune response in this context, indicating a need for a source of NKT cells that could be used to supplement the endogenous cell population. Induced pluripotent stem cells (iPSCs) hold tremendous potential for cell-replacement therapy, but whether it is possible to generate functionally competent NKT cells from iPSCs has not been rigorously assessed. In this study, we successfully derived iPSCs both from embryonic fibroblasts from mice harboring functional NKT cell–specific rearranged T cell receptor loci in the germline and from splenic NKT cells from WT adult mice. These iPSCs could be differentiated into NKT cells in vitro and secreted large amounts of the Th1 cytokine IFN-γ. Importantly, iPSC-derived NKT cells recapitulated the known adjuvant effects of natural NKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent NKT cells via an iPSC phase, an approach that may be adapted for NKT cell–targeted therapy in humans.     

HLA-DR+ leukocytes acquire CD1 antigens in embryonic and fetal human skin and contain functional antigen-presenting cells

Adequate numbers and functional maturity are needed for leukocytes to exhibit a protective role in host defense. During intrauterine life, the skin immune system has to acquire these prerequisites to protect the newborn from infection in the hostile external environment after birth. We investigated the quantitative, phenotypic, and functional development of skin leukocytes and analyzed the factors controlling their proliferation and trafficking during skin development. We show that CD45⁺ leukocytes are scattered in embryonic human skin and that their numbers continuously increase as the developing skin generates an environment that promotes proliferation of skin resident leukocytes as well as the influx of leukocytes from the circulation. We also found that CD45⁺HLA-DR^highCD1c⁺ dendritic cells (DCs) are already present in the epidermis and dermis at 9 wk estimated gestational age (EGA) and that transforming growth factor β1 production precedes Langerin and CD1a expression on CD45⁺CD1c⁺ Langerhans cell (LC) precursors. Functionally, embryonic antigen-presenting cells (APCs) are able to phagocytose antigen, to up-regulate costimulatory molecules upon culture, and to efficiently stimulate T cells in a mixed lymphocyte reaction. Collectively, our data provide insight into skin DC biology and the mechanisms through which skin DCs presumably populate the skin during development.     

Thymus-homing peripheral dendritic cells constitute two of the three major subsets of dendritic cells in the steady-state thymus

Many dendritic cells (DCs) in the normal mouse thymus are generated intrathymically from common T cell/DC progenitors. However, our previous work suggested that at least 50% of thymic DCs originate independently of these progenitors. We now formally demonstrate by parabiotic, adoptive transfer, and developmental studies that two of the three major subsets of thymic DCs originate extrathymically and continually migrate to the thymus, where they occupy a finite number of microenvironmental niches. The thymus-homing DCs consisted of immature plasmacytoid DCs (pDCs) and the signal regulatory protein α–positive (Sirpα⁺) CD11b⁺ CD8α⁻ subset of conventional DCs (cDCs), both of which could take up and transport circulating antigen to the thymus. The cDCs of intrathymic origin were mostly Sirpα⁻ CD11b⁻ CD8α^hi cells. Upon arrival in the thymus, the migrant pDCs enlarged and up-regulated CD11c, major histocompatibility complex II (MHC II), and CD8α, but maintained their plasmacytoid morphology. In contrast, the migrant cDCs proliferated extensively, up-regulated CD11c, MHC II, and CD86, and expressed dendritic processes. The possible functional implications of these findings are discussed.Three major subsets of DCs have been identified in the mouse and human thymus (1). The most intensively studied subset in the mouse has a signal regulatory protein α–negative (Sirpα⁻) CD11b⁻ CD8α^hi phenotype and is thought to arise intrathymically from a common T cell/DC precursor. These conventional DCs (cDCs) are generally presumed to be involved in the negative selection of developing thymocytes. In contrast, the origins and in vivo functions of the two other subsets of thymic DCs are unknown. One subset is composed of plasmacytoid DCs (pDCs) and the other is composed of Sirpα⁺ CD11b⁺ CD8α⁻ cDCs.In previous parabiotic and adoptive transfer experiments, we provided evidence that at least 50% of the DCs in the steady-state thymus developed independently of thymocyte progenitors (2). These included pDCs and possibly the subset of CD11b⁺ CD8α⁻ cDCs. In contrast, most of the DCs whose generation was linked to the importation of thymocyte progenitors were CD11b⁻ CD8α^hi cDCs, as also suggested by the experiments of Porritt et al. (3). These results raised the possibility that two of the three subsets of thymic DCs might be formed extrathymically.In studies involving the induction of immune deviation (Th1 to Th2 polarization) in mice injected intraocularly with antigen, we detected a population of F4/80⁺ antigen-presenting cells that migrated to the thymus and activated NKT reg cells (unpublished data) (4, 5). We also observed that self–MHC II–specific CD4⁺ CD25⁺ T reg cells appeared in the thymus of post–cyclosporin A (CSA)–treated mice at the time when an influx of tolerogenic DCs from the periphery occurs (6–8). In addition, abundant evidence from other laboratories has demonstrated that thymus-homing inflammatory DCs can induce both dominant and deletional tolerance to peripheral self- and nonself-antigens (see Discussion). These observations led us to hypothesize that, in addition to the promiscuous intrathymic expression of tissue-restricted antigens (9), the migration of DCs from the blood to thymus constituted a major pathway by which the induction of dominant and deletional thymic tolerance to peripheral self- and nonself-antigens may also occur under steady-state conditions (2, 10).Although the present study does not formally test this hypothesis, it does provide supporting data by demonstrating that (a) most, if not all, of the pDCs and approximately one third of the cDCs (those expressing Sirpα and CD11b) in the steady-state thymus are recent emigrants from the blood; (b) immature, noninflammatory, thymus-homing pDCs and cDCs can take up both soluble and particulate circulating antigen and transport it to the thymus; (c) once in the thymus, the pDCs enlarge and assume a semimature phenotype by up-regulating CD11c, MHC II, and CD8α (but not CD86) while maintaining a plasmacytoid morphology; and (d) the thymus-homing Sirpα⁺ CD11b⁺ cDCs proliferate extensively in the thymus, up-regulate CD86 as well as CD11c and MHC II, and assume a mature DC morphology. Some of the functional implications of these findings have recently been reported by Proietto et al. (11), who observed that the Sirpα⁺ CD11b⁺ subset of thymic cDCs could induce CD4⁺ CD25⁺ Foxp3⁺ T reg cells from CD4⁺ CD25⁻ Foxp3⁻ thymocytes in vitro, and that thymus-homing DCs could induce antigen-specific (OVA) deletion and T reg cell formation in transplanted thymus lobes in vivo. Given the propensity of semimature pDCs to induce T reg cells in the periphery (12–14), it is possible that they too may contribute to the induction of central tolerance.     

Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus

DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime–modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase 3 (Flt3)–ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8⁺ interferon (IFN) γ⁺ T cells and polyfunctional CD8⁺ T cells producing IFN-γ, tumor necrosis factor α, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac₂₅₁ and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL.     

CD40 Ligand Is Not Essential for Induction of Type 1 Cytokine Responses or Protective Immunity after Primary or Secondary Infection With Histoplasma capsulatum

The induction of type 1 immune responses (interleukin [IL]-12, interferon [IFN]-γ) has been shown to be important in mediating protection against many intracellular infections including Histoplasma capsulatum. Costimulatory molecules such as CD40 ligand (CD40L) have been shown to be a central regulator of type 1 responses in vivo. To study the role of CD40L in mediating protection against infection with H. capsulatum, CD40L-deficient (CD40L^−/−) and CD40L^+/+ mice were infected with H. capsulatum and assessed for various parameters. After a lethal challenge of H. capsulatum, CD40L^−/− mice were not substantially different from CD40L^+/+ mice in terms of mortality, fungal burden, or production of IFN-γ, IL-12, nitric oxide, or tumor necrosis factor α. Moreover, CD40L^−/− mice treated with anti–IFN-γ or anti–IL-12 at the time of infection had accelerated mortality, providing further evidence that IL-12 and IFN-γ are produced in vivo in the absence of CD40L. In addition, CD40L^−/− mice infected with a sublethal dose of H. capsulatum survived infection, whereas all mice infected with the same dose and treated with anti–IFN-γ had accelerated mortality, demonstrating that IFN-γ but not CD40L was essential for primary immunity to H. capsulatum infection. Interestingly, depletion of either CD4⁺ or CD8⁺ T cells resulted in accelerated mortality in CD40L^−/− mice, suggesting a critical role for these cells in response to infection. Finally, CD40L^−/− mice initially infected with a sublethal dose of H. capsulatum were protected from secondary infection with a lethal dose of H. capsulatum, demonstrating that CD40L is not required for the maintenance of memory immunity.     

In Vivo Protection Provided by a Synthetic New Alpha-Galactosyl Ceramide Analog against Bacterial and Viral Infections in Murine Models

Alpha-galactosyl ceramide (α-GalCer) has been known to bind to the CD1d receptor on dendritic cells and activate invariant natural killer T (iNKT) cells, which subsequently secrete T-helper-cell 1 (Th1) and Th2 cytokines, which correlate with anti-infection activity and the prevention of autoimmune diseases, respectively. α-GalCer elicits the secretion of these two cytokines nonselectively, and thus, its effectiveness is limited by the opposing effects of the Th1 and Th2 cytokines. Reported here is the synthesis of a new α-GalCer analog (compound C34), based on the structure of CD1d, with a 4-(4-fluorophenoxy) phenyl undecanoyl modification of the N-acyl moiety of α-GalCer. Using several murine bacterial and viral infection models, we demonstrated that C34 has superior antibacterial and antiviral activities in comparison with those of several other Th1-selective glycolipids and that it is most effective by administering it to mice in a prophylactic manner before or shortly after infection.Natural killer T (NKT) cells contribute to a variety of immunological processes through the recognition of NKT cell receptors by lipid and glycolipid antigens presented by CD1d molecules (2, 35). CD1d molecules are major histocompatibility complex class I-like proteins and are expressed by most monocytes, macrophages, dendritic cells, and B cells as well as by nonlymphoid cells. CD1d presents glycolipid antigens to CD1d-restricted T cells (or NKT cells), which are implicated in the host innate defense system through the production of T-helper-cell 1 (Th1) and Th2 types of cytokines, such as gamma interferon (IFN-γ) and interleukin-4 (IL-4), respectively (32, 39). The production of Th1 cytokines is shown to correlate with the antitumor, antibacterial, and antiviral effects of glycolipids, while the ability to induce the Th2 cytokines is thought to correlate with the amelioration of certain autoimmune diseases, such as type 1 diabetes (9, 10, 28).Among the variety of ligands that bind to CD1d, the most well studied one is alpha-galactosyl ceramide (α-GalCer) (7, 17, 30), which is a synthetic, structurally optimized compound of the natural product lead identified from the marine sponge (Agelas mauritianus) as agelasphin-9b (24). The synthetic analog (2S,3S,4R)-1-O-(α-d-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol was made as KRN7000 at Kirin Brewery, Japan (23), and has generally been referred to as α-GalCer. Mice treated with α-GalCer were shown to be protected against a variety of infections (13, 15, 16, 22, 33). However, the effectiveness of α-GalCer is limited by the opposing effects of the Th1 and Th2 cytokines induced by this glycolipid. In order to develop selective Th1 or Th2 activators, many α-GalCer analogs were synthesized (1, 3, 5, 8, 12, 14, 21, 34, 36), and their immune-modulating activities were shown to be related to the affinity of binding to CD1d (8). Recent studies using a CD1d array established that the secretion of IFN-γ and IL-4 by NKT cells is determined by the binding of α-GalCer analogs to CD1d (21), and a higher affinity for CD1d would shift the cytokine release profile toward a stronger Th1 response (3, 21).This report described the synthesis of the 4-(4-fluorophenoxy)phenyl undecanoyl derivative of α-GalCer as a selective Th1 activator. This analog was designed on the basis of the structure of CD1d, where the N-acyl group of the glycolipid interacts most favorably with the lipid binding moiety of CD1d (36). Its efficacies for protection against both bacterial and viral infections were compared with those of α-GalCer and several other analogs using murine models of infection. Our study found that among the synthetic α-GalCer analogs tested, compound C34 is the most effective in offering protection against all of the bacterial and viral infection models tested, especially when it is given in a prophylactic manner or shortly after infection.     

Vα24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages

Tumor infiltration with Vα24-invariant NKT cells (NKTs) associates with favorable outcome in neuroblastoma and other cancers. Although NKTs can be directly cytotoxic against CD1d⁺ cells, the majority of human tumors are CD1d^–. Therefore, the role of NKTs in cancer remains largely unknown. Here, we demonstrate that CD68⁺ tumor-associated monocytes/macrophages (TAMs) represented the majority of CD1d-expressing cells in primary human neuroblastomas. TAMs stimulated neuroblastoma growth in human cell lines and their xenografts in NOD/SCID mice via IL-6 production. Indeed, TAMs produced IL-6 in primary tumors and in the BM of patients with metastatic neuroblastoma. Gene expression analysis using TaqMan low-density arrays of 129 primary human neuroblastomas without MYCN amplification revealed that high-level expression of TAM-specific genes (CD14, CD16, IL6, IL6R, and TGFB1) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing of monocytes was CD1d restricted because it was inhibited by a CD1d-specific mAb. Cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and suppressed tumor growth compared with mice transferred with monocytes alone. Thus, killing of TAMs reveals what we believe to be a novel mechanism of NKT antitumor activity that relates to the disease outcome.     

Phenotypic and Functional Separation of Memory and Effector Human CD8+ T Cells

Human CD8⁺ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8⁺ T cell subset. First, CD45RA⁻CD45R0⁺ cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8⁺ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon γ, tumor necrosis factor α, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8⁺CD45RA⁺CD27⁻ population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-γ and TNF-α. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8⁺CD45RA⁺CD27⁻ cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8⁺ T cell subset.     

Identification of a Committed T Cell Precursor Population in Adult Human Peripheral Blood

Here, we report data concerning the discovery in adult human peripheral blood of a precursor cell population able to differentiate into CD4⁺CD3⁺αβ⁺ mature T cells. These cells, which represent 0.1–0.5% of total peripheral blood mononuclear cells (PBMC), express substantial levels of CD4, but lack CD3 surface expression. At a molecular level, they express the pre-T cell receptor α (pTα) gene, CD3-γ, CD-δ and CD-ε, and RAG-1 recombination enzyme and have initiated rearrangements in the T cell receptor (TCR)-β locus (D–J). Moreover, low levels of CD3ε protein, but not of TCR-β chain, can be detected in their cytoplasm. Our results suggest that CD4⁺CD3⁻ cells identified in peripheral blood are different from CD3⁻CD4⁺CD8⁻ thymocytes and may contain precursors of an extrathymic T cell differentiation pathway.     

The Thymus Contains a High Frequency of Cells that Prevent Autoimmune Diabetes on Transfer into Prediabetic Recipients

Rats of the PVG.RT1^u strain develop autoimmune diabetes when thymectomized at 6 wk of age and are rendered relatively lymphopenic by a cumulative dose of 1,000 rads ¹³⁷Cs γ-irradiation given in four split doses. Previous studies have shown that the disease is prevented by the intravenous injection of 5 × 10⁶ CD4⁺ CD45RC⁻ TCRαβ⁺ RT6⁺ peripheral T cells from normal syngeneic donors. These cells have a memory phenotype and are presumably primed to some extrathymic antigen. However, we now report that the CD4⁺ CD8⁻ population of mature thymocytes is a very potent source of cells, with the capacity to prevent diabetes in our lymphopenic animals. As few as 6 × 10⁵ of these cells protect ~50% of recipients and the level of protection increases with cell dose. It appears that one characteristic of the intrathymic selection of the T cell repertoire is the generation of cells that regulate the autoimmune potential of peripheral T cells that have been neither clonally deleted intrathymically nor rendered irreversibly anergic in the periphery.