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Ultraviolet (UV) wavelengths in sunlight are the prime cause of skin cancer in humans with both the UVA and UVB wavebands making a contribution to photocarcinogenesis. UV has many different biological effects on the skin that contribute to carcinogenesis, including suppression of adaptive immunity, sunburn and altering the migration of mast cells into and away from irradiated skin. Many molecular mechanisms have been identified as contributing to skin responses to UV. Recently, using gene set enrichment analysis of microarray data, we identified the alternative complement pathway with a central role for factor B (fB) in UVA-induced immunosuppression. In the current study we used mice genetically deficient in fB (fB-/- mice) to study the functional role of the alternative complement pathway in skin responses to UV. We found that fB is required for not only UVA but also UVB-induced immunosuppression and solar-simulated UV induction of the oedemal component of sunburn. Factor B-/- mice had a larger number of resident skin mast cells than control mice, but unlike the controls did not respond to UV by increasing mast cell infiltration into the skin. This study provides evidence for a function role for fB in skin responses to UV radiation. Factor B regulates UVA and UVB induced immunosuppression, UV induced oedema and mast cell infiltration into the skin. The alternative complement pathway is therefore an important regulator of skin responses to UV.

Mouse natural killer T (NKT) cells with an invariant V alpha14-J alpha18 rearrangement (V alpha14 invariant [V alpha14i] NKT cells) are either CD4(+)CD8(-) or CD4(-)CD8(-). Because transgenic mice with forced CD8 expression in all T cells exhibited a profound NKT cell deficit, the absence of CD8 has been attributed to negative selection. We now present evidence that CD8 does not serve as a coreceptor for CD1d recognition and that the defect in development in CD8 transgene homozygous mice is the result of a reduction in secondary T cell receptor alpha rearrangements. Thymocytes from mice hemizygous for the CD8 transgene have a less severe rearrangement defect and have functional CD8(+) V alpha14i NKT cells. Furthermore, we demonstrate that the transcription factor Th, Poxviruses and Zinc finger, and Krüppel family (Th-POK) is expressed by V alpha14i NKT cells throughout their differentiation and is necessary both to silence CD8 expression and for the functional maturity of V alpha14i NKT cells. We therefore suggest that Th-POK expression is required for the normal development of V alpha14i NKT cells and that the absence of CD8 expression by these cells is a by-product of such expression, as opposed to the result of negative selection of CD8-expressing V alpha14i NKT cells.

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR α-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d−/− mice that are completely deficient for NKT cells. CD1d tetramer− CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer− CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.

Natural killer (NK) T cells with an invariant Vα14 rearrangement (Vα14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid α-galactosyl ceramide (α-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Vα14iT cell antigen receptor (TCR) has a high affinity for the α-GalCer/CD1d complex, driven by a long half-life (t1/2). Although this result could have reflected the unique attributes of α-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Vα14iNKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a longt1/2. Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Vα14iNKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Vα14iNKT cells.

Natural killer T (NKT) cells are a unique lymphocyte subtype implicated in the regulation of autoimmunity, particularly diabetes and experimental allergic encephalomyelitis in animal models. In some reports, NKT-cell regulation was revealed only following vigorous activation by a synthetic glycolipid, a process that might not occur naturally. Patients with diverse autoimmune diseases have reduced NKT-cell counts and, in diabetes and multiple sclerosis, effective NKT-cell regulation correlates with the secretion of Th2 cytokines. Although current controversy surrounds the importance of NKT cells and their modes of action, they represent a potentially important clinical target.

NKT cells are enigmatic lymphocytes that respond to glycolipid Ags presented by CD1d. Although they are key immunoregulatory cells, with a critical role in immunity to cancer, infection, and autoimmune diseases, little is known about how they respond to antigenic challenge. Current theories suggest that NKT cells die within hours of stimulation, implying that their direct impact on the immune system derives from the initial cytokine burst released before their death. Here we show that NKT cell disappearance results from TCR down-regulation rather than apoptosis, and that they expand to many times their normal number in peripheral tissues within 2–3 days of stimulation, before contracting to normal numbers over subsequent days. This expansion is associated with ongoing cytokine production, biased toward a Th1 (IFN-γ+ IL-4−) phenotype, in contrast to their initial Th0 (IFN-γ+IL-4+) phenotype. This study provides critical new insight into how NKT cells can have such a major impact on immune responses, lasting many days beyond the initial stimulation of these cells.

A defect in RelB, a member of the Rel/nuclear factor (NF)-κB family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-κB–inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/− × aly/+ compound heterozygous mice. After stimulation with α-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/− or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.

NKT cells represent a unique T cell lineage that recognize glycolipid antigens in the context of the non-classical MHC class I molecule CD1d. NKT cells are potent producers of immunoregulatory cytokines, and have been implicated in several different autoimmune diseases in mice and humans, including Type 1 diabetes, experimental autoimmune encephalomyelitis--a mouse model for multiple sclerosis, systemic lupus erythematosus, and scleroderma. This review will cover the evidence for an involvement for NKT cells in these autoimmune diseases, and discuss the potential for therapeutic manipulation of these cells as a means of preventing autoimmune disease in the clinic.

The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/alpha-galactosylceramide (CD1d/alphaGC) tetramers and anti-NK1.1 to investigate NKT cell development in vitro and in vivo. Confirming the thymus-dependence of these cells, we show that CD1d/alphaGC tetramer-binding NKT cells, including NK1.1(+) and NK1.1(-) subsets, develop in fetal thymus organ culture (FTOC) and are completely absent in nude mice. Ontogenically, CD1d/alphaGC tetramer-binding NKT cells first appear in the thymus, at day 5 after birth, as CD4(+)CD8(-)NK1.1(-)cells. NK1.1(+) NKT cells, including CD4(+) and CD4(-)CD8(-) subsets, appeared at days 7-8 but remained a minor subset until at least 3 wk of age. Using intrathymic transfer experiments, CD4(+)NK1.1(-) NKT cells gave rise to NK1.1(+) NKT cells (including CD4(+) and CD4(-) subsets), but not vice-versa. This maturation step was not required for NKT cells to migrate to other tissues, as NK1.1(-) NKT cells were detected in liver and spleen as early as day 8 after birth, and the majority of NKT cells among recent thymic emigrants (RTE) were NK1.1(-). Further elucidation of this NKT cell developmental pathway should prove to be invaluable for studying the mechanisms that regulate the development of these cells.

CD1d-restricted Vα14-Jα281 invariant αβTCR+ (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as αβTCR+CD4−CD8− and DX5+CD3+ have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/α-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5+CD3+) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4+ subset and particularly in NK1.1-congenic BALB/c mice.

The proposed roles of NK1.1(+) T (NKT) cells in immune responses range from suppression of autoimmunity to tumor rejection. Heterogeneity of these cells contributes to the controversy surrounding their development and function. This review aims to provide an update on NKT cell biology and, whenever possible, to compare what is known about NKT-cell subsets.

NK1.1(+)alpha betaTCR(+) (NKT) cells have several important roles including tumor rejection and prevention of autoimmune disease. Although both CD4(+) and CD4(-)CD8(-) double-negative (DN) subsets of NKT cells have been identified, they are usually described as one population. Here, we show that NKT cells are phenotypically, functionally and developmentally heterogeneous, and that three distinct subsets (CD4(+), DN and CD8(+)) are differentially distributed in a tissue-specific fashion. CD8(+) NKT cells are present in all tissues but the thymus, and are highly enriched for CD8alpha(+)beta(-) cells. These subsets differ in their expression of a range of cell surface molecules (Vbeta8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL-4 and IFN-gamma, with splenic NKT cell subsets producing lower levels than thymic NKT cells. Developmentally, most CD4(+) and DN NKT cells are thymus dependent, in contrast to CD8(+) NKT cells, and are also present amongst recent thymic emigrants in spleen and liver. TCR Jalpha281-deficient mice show a dramatic deficiency in thymic NKT cells, whereas a significant NKT cell population (enriched for the DN and CD8(+) subsets) is still present in the periphery. Taken together, this study reveals a far greater level of complexity within the NKT cell population than previously recognized.

Neonatal thymectomy of mice 3 days after birth but not at birth leads to T cell-mediated, organ-specific, autoimmune disease in a strain-dependent manner. The mechanisms that lead to disease in this model remain unknown, but the answer may lie in a deficiency of thymus-dependent cells or factors. One candidate is the relatively rare population of NK1.1 + T cells (NKT cells). Conventional alphabetaTCR+ T cells appear in the thymus from days 17-18 of embryogenesis and start emigrating to the periphery around birth, whereas the development of NKT cells is thought to be delayed until at least 1 week after birth. We have confirmed this to be the case in both (BALB/c x C57BL/6)F1 (autoimmune susceptible) and C57BL/6 (autoimmune resistant) mice. Moreover, examination of T cells (in spleen, lymph nodes, liver and bone marrow) from mice following 3 day neonatal thymectomy revealed a significant reduction in the presence of NKT cells in all tissues. However, the extent of depletion was generally more pronounced in (BALB/c x C57BL/6)F1 than in C57BL/6 mice, and the few remaining NKT cells in C57BL/6 mice were enriched for a CD4-CD8int subset which is absent from the thymus and may represent a distinct lineage of thymus-independent NKT cells. Given mounting evidence of a role for NKT cells in protection from autoimmune disease, it is possible that their specific removal by neonatal thymectomy may contribute to the susceptibility of these mice to autoimmune disease.

Natural killer (NK) T cells using an invariant Vα14 (Vα14i) T cell receptor rearrangement form a distinct immunoregulatory T cell lineage. Several studies indicated that a NK1.1−Vα14iNKT precursor cell differentiates and expands within the thymus before export to the peripheral tissues occurs. However, little is known about the signals that cause the emigration of Vα14iNKT cells from the thymus to the periphery. Here we show that signaling of lymphotoxin (LT) αβ through the LTβ receptor (LTβR) is indispensable for regulating peripheral but not thymic Vα14iNKT cell numbers. Homing to and homeostatic proliferation of thymic Vα14iNKT cells in peripheral organs, however, was not dependent on LTβR. Instead, our data indicate that a LTβR-expressing thymic stromal cell regulates the thymic emigration of Vα14iNKT cells but not conventional T cell receptor αβ cells.

NKT cell development takes place in the thymus, beginning when these cells branch away from CD4+CD8+ mainstream thymocytes upon expression of the Valpha14Jalpha18 T cell receptor (TCR) and recognition of the CD1d molecule. Although NKT cells express an invariant TCR alpha chain, the diverse TCR beta expression leaves open the possibility that the development of these cells is shaped by glycolipid antigen recognition in the context of CD1d. Here, we show that the presence of an agonist glycolipid ligand, alpha-galactosylceramide, while NKT cells are developing in vitro or in vivo, specifically ablates their development. In contrast, the delayed introduction of this compound in vitro or in vivo, after NKT cells have developed, does not deplete these cells. These data indicate that NKT cells pass through a developmental window where they are susceptible to TCR-mediated negative selection, and suggest that NKT cells with a potentially high level of self reactivity can be removed from the NKT cell repertoire before they exit the thymus.

NOD mice develop spontaneous IDDM as a result of T-cell-mediated autoimmune destruction of pancreatic beta-cells. It is not known why these T-cells become autoreactive, nor is it clear whether the breakdown in self-tolerance reflects a general problem in T-cell development or a selective defect in an as yet undefined regulatory cell population. In this study, we showed that NOD mice, although relatively normal with regard to most thymocyte subsets, exhibit a marked deficiency in alphabetaTCR+CD4-CD8- (alphabeta+DN) T-cells in the thymus and, to a lesser extent, in the periphery. These T-cells have been termed NKT cells (NK1.1+-like T-cells) because they share some cell surface markers with conventional natural killer (NK) cells. To examine the role of these cells in the pathogenesis of IDDM, semiallogeneic or syngeneic double-negative (DN) thymocytes, enriched for NKT cells, were transferred into intact 4-week-old NOD recipients; the onset of diabetes was then monitored over the ensuing 30 weeks. Mice receiving NKT-enriched thymocytes did not develop diabetes, whereas mice receiving unfractionated thymocytes or phosphate-buffered saline developed diabetes at the normal rate. NKT cells represent a distinct T-cell lineage that has been shown to play a role in immunoregulation in vivo. The deficiency of these cells observed in NOD mice may therefore contribute to destruction of pancreatic islet cells by conventional T-cells.