Kirsten J. L. Hammond

NKT cells: facts, functions and fallacies

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.

A Natural Killer T (NKT) Cell Developmental Pathway Involving a Thymus-dependent NK1.1−CD4+ CD1d-dependent Precursor Stage

The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/α-galactosylceramide (CD1d/αGC) 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/αGC 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/αGC 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.

NKT cells are phenotypically and functionally diverse

NK1.1+α βTCR+ (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 CD8α+β– cells. These subsets differ in their expression of a range of cell surface molecules (Vβ8, DX5, CD69, CD45RB, Ly6C) and in their ability to produce IL‐4 and IFN‐γ, 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 Jα281‐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.

Glycolipid Antigen Drives Rapid Expansion and Sustained Cytokine Production by NK T Cells

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.

CD1d-Restricted NKT Cells: An Interstrain Comparison

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.

Association Between αβTCR+CD4−CD8− T-Cell Deficiency and IDDM in NOD/Lt Mice

NOD mice develop spontaneous IDDM as a result of T-cell–mediated autoimmune destruction of pancreatic β-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 αβTCR+CD4−CD8− (αβ+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.

Differential Requirement for Rel/Nuclear Factor κB Family Members in Natural Killer T Cell Development

Natural killer T (NKT) cells have been implicated in diverse immune responses ranging from suppression of autoimmunity to tumor rejection. Thymus-dependent NKT cells are positively selected by the major histocompatibility complex class I–like molecule CD1d, but the molecular events downstream of CD1d are still poorly understood. Here, we show that distinct members of the Rel/nuclear factor (NF)-κB family of transcription factors were required in both hematopoietic and nonhematopoietic cells for normal development of thymic NKT cells. Activation of NF-κB via the classical IκBα-regulated pathway was required in a cell autonomous manner for the transition of NK-1.1–negative precursors that express the TCR Vα14-Jα18 chain to mature NK-1.1–positive NKT cells. The Rel/NF-κB family member RelB, on the other hand, had to be expressed in radiation resistant thymic stromal cells for the generation of early NK-1.1–negative NKT precursors. Moreover, NF-κB–inducing kinase (NIK) was required for both constitutive thymic DNA binding of RelB and the specific induction of RelB complexes in vitro. Thus, distinct Rel/NF-κB family members in hematopoietic and nonhematopoietic cells regulate NKT cell development with a unique requirement for NIK-mediated activation of RelB in thymic stroma.

NIK-dependent RelB Activation Defines a Unique Signaling Pathway for the Development of Vα14i NKT 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.

Intrathymic NKT cell development is blocked by the presence of α‐galactosylceramide

NKT cell development takes place in the thymus, beginning when these cells branch away from CD4+CD8+ mainstream thymocytes upon expression of the Vα14Jα18 T cell receptor (TCR) and recognition of the CD1d molecule. Although NKT cells express an invariant TCR α chain, the diverse TCR β 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, α‐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.

DX5/CD49b-positive T cells are not synonymous with CD1d-dependent 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.