Jennifer L. Matsuda

NKT cells derive from double-positive thymocytes that are positively selected by CD1d

CD1d-reactive NKT cells are a separate T cell sublineage. Instructive models propose that NKT cells branch off the mainstream developmental pathway because of their T cell antigen receptor specificity, whereas stochastic models would propose that they develop from precursor cells committed to this sublineage before variable-gene rearrangement. We show here that immature double-positive (DP) thymocytes form the canonical rearranged Vα gene of NKT cells at nearly equivalent frequencies in the presence or absence of CD1d expression. After interacting with CD1d in the thymus, these cells give rise to expanded populations of NKT cells—including both CD4+ and double-negative lymphocytes in the thymus and periphery—that express this α chain. These results confirm the existence of a DP intermediate for CD1d-reactive NKT cells. They also show that the early developmental stages of these T cells are not governed by a distinct mechanism, which is consistent with the TCR-instructive model of differentiation.

Homeostasis of V alpha 14i NKT cells.

CD1d-reactive natural killer T (NKT) cells with an invariant Vα14 rearrangement (Vα14i) are a distinct subset of T lymphocytes that likely have important immune-regulatory functions. Little is known regarding the factors responsible for their peripheral survival. Using α-galactosylceramide–containing CD1d tetramers to detect Vα14i NKT cells, we show here that the expansion of Vα14i NKT cells in lymphopenic mice was not dependent on CD1d expression and was unaffected by the presence of host NKT cells. Additionally, we found that IL-15 was important in the expansion and/or survival of Vα14i NKT cells, with IL-7 playing a lesser role. These results demonstrate that the homeostatic requirements for CD1d-restricted NKT cells, which are CD4+ or CD4−CD8−, resemble those of CD8+ memory T cells. We propose that this expansion and/or survival in the periphery of Vα14i NKT cells is affected by competition for IL-15, and that IL-15–requiring cells—such as NK cells and CD8+ memory cells—may define the Vα14i NKT cell niche.

CD1d-expressing Dendritic Cells but Not Thymic Epithelial Cells Can Mediate Negative Selection of NKT Cells

Natural killer T (NKT) cells are a unique immunoregulatory T cell population that is positively selected by CD1d-expressing thymocytes. Previous studies have shown that NKT cells exhibit autoreactivity, which raises the question of whether they are subject to negative selection. Here, we report that the addition of agonist glycolipid α-galactosylceramide (α-GalCer) to a fetal thymic organ culture (FTOC) induces a dose-dependent disappearance of NKT cells, suggesting that NKT cells are susceptible to negative selection. Overexpression of CD1d in transgenic (Tg) mice results in reduced numbers of NKT cells, and the residual NKT cells in CD1d-Tg mice exhibit both an altered Vβ usage and a reduced sensitivity to antigen. Furthermore, bone marrow (BM) chimeras between Tg and WT mice reveal that CD1d-expressing BM-derived dendritic cells, but not thymic epithelial cells, mediate the efficient negative selection of NKT cells. Thus, our data suggest that NKT cells developmentally undergo negative selection when engaged by high-avidity antigen or abundant self-antigen.

Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor β repertoire and small clone size

CD1d-restricted natural killer (NK) T cells reactive with the glycolipid α-galactosylceramide (α-GalCer) are a distinct lymphocyte sublineage. They express an invariant Vα14-Jα18 T cell receptor (TcR), but the role of the β chain has been controversial. Here, we have used CD1d tetramers to identify and isolate NK T cells based on their antigen specificity. In mice lacking germline Vβ8, most of the α-GalCer-reactive T cells express either Vβ2 or Vβ7, strong Vβ selection being revealed by the lack of an increase in other Vβ regions. By contrast to the selection for complementarity determining region (CDR) 3β sequences in some anti-peptide responses, α-GalCer-reactive T cells have polyclonal CDR3β sequences. There is little CDR3β sequence redundancy between organs or individual mice, and, surprisingly, there also is no evidence for organ-specific CDR3β sequence motifs. These data argue against a T cell receptor-mediated self-reactivity for tissue-specific CD1d-bound ligands. Each NKT clone is represented by only 5–10 cells. This clone size is similar to naive conventional T cells, and much lower than that reported for memory T cells, although NK T cells have an activated/memory phenotype.

Homeostasis of Vα14i NKT cells

CD1d-reactive natural killer T (NKT) cells with an invariant Vα14 rearrangement (Vα14i) are a distinct subset of T lymphocytes that likely have important immune-regulatory functions. Little is known regarding the factors responsible for their peripheral survival. Using α-galactosylceramide–containing CD1d tetramers to detect Vα14i NKT cells, we show here that the expansion of Vα14i NKT cells in lymphopenic mice was not dependent on CD1d expression and was unaffected by the presence of host NKT cells. Additionally, we found that IL-15 was important in the expansion and/or survival of Vα14i NKT cells, with IL-7 playing a lesser role. These results demonstrate that the homeostatic requirements for CD1d-restricted NKT cells, which are CD4+ or CD4−CD8−, resemble those of CD8+ memory T cells. We propose that this expansion and/or survival in the periphery of Vα14i NKT cells is affected by competition for IL-15, and that IL-15–requiring cells—such as NK cells and CD8+ memory cells—may define the Vα14i NKT cell niche.

Membrane Lymphotoxin Is Required for the Development of Different Subpopulations of NK T Cells

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTα and LTβ, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTα−/− and LTβ−/− mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTα−/− and LTβ−/− mice did not respond to the lipoglycan α-galactosylceramide, which is presented by mouse CD1 to Vα14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and α-galactosylceramide reactive and those that are not, were affected by disruption of the LTα and LTβ genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTβ receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Lower TCR repertoire diversity in Traj18-deficient mice.

705 during infection with bacterial, viral, protozoan and fungal pathogens2. Because iNKT cells are highly conserved in mice and humans3, mouse models of deficiency in iNKT cells represent useful tools for immunologists. Two similar but not equivalent models of deficiency in iNKT cells exist. One makes use of mice deficient in CD1d (Cd1d1−/−Cd1d2−/− mice)4, which prevents the development of any CD1d-reactive T cells, including iNKT cells. Another model directly targets Traj18 (which encodes the T cell antigen receptor (TCR) a-chain joining region 18 (Ja18)), which in combination with Trav11 (which encodes the TCR a-chain variable region 14 (Va14)) is absolutely required for formation of an iNKT TCR with the appropriate antigenic specificity6. The RAG-1 and RAG-2 recombinases drive successive rearrangement of genes encoding TCR band a-chains during thymocyte development. To the Editor: Natural killer T cells (NKT cells) constitute a distinct subset of T lymphocytes that can modulate immune responses through the rapid release of cytokines and direct interactions with other cells of the immune system1. Thus, NKT cells serve as an important link between the innate and adaptive immune systems and are promising targets for immunotherapy. Type I NKT cells (iNKT cells) are the most prevalent NKT cells in mice and have similar properties in mice and humans. The iNKT cells have evolved to recognize lipid-based antigens presented by the nonclassical major histocompatibility complex (MHC)-like molecule CD1d. Many studies of humans and mice have reported a strong association between defects in iNKT cells and greater susceptibility to autoimmune disease and cancer. In addition, iNKT cells are known to have important roles Lower TCR repertoire diversity in Traj18-deficient mice

Mutation of the Traj18 gene segment using TALENs to generate Natural Killer T cell deficient mice

Invariant Natural Killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKT cells express T cell antigen receptors (TCRs) comprising a unique TCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain Trbv TCRβ chains, these TCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT deficiency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specificity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-deficient mouse strain using transcription activator-like effector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confirm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional T cells, regulatory T cells, and type Ib NKT cells is normal. This new mouse strain will serve as a new model of iNKT cell deficiency to facilitate our understanding of iNKT biology.

Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors

Significance The evolutionary hypothesis for T-cell antigen receptor–peptide major histocompatibility complex (TCR–pMHC) interaction posits the existence of germ-line–encoded rules by which the TCR is biased toward recognition of the MHC. Understanding these rules is important for our knowledge of how to manipulate this important interaction at the center of adaptive immunity. In this study, we highlight the flexibility of thymic selection as well as the existence of these rules by generating knockin mutant MHC mice and extensively studying the TCR repertoires of T cells selected on the mutant MHC molecules. Identifying novel TCR subfamilies that are most evolutionarily conserved to recognize specific areas of the MHC is the first step in advancing our knowledge of this central interaction. The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

A novel mouse model of conditional IRAK-M deficiency in myeloid cells: application in lung Pseudomonas aeruginosa infection

Myeloid cells such as macrophages are critical to innate defense against infection. IL-1 receptor-associated kinase M (IRAK-M) is a negative regulator of TLR signaling during bacterial infection, but the role of myeloid cell IRAK-M in bacterial infection is unclear. Our goal was to generate a novel conditional knockout mouse model to define the role of myeloid cell IRAK-M during bacterial infection. Myeloid cell-specific IRAK-M knockout mice were generated by crossing IRAK-M floxed mice with LysM–Cre knock-in mice. The resulting LysM–Cre+/IRAK-Mfl/wt and control (LysM–Cre–/IRAK-Mfl/wt) mice were intranasally infected with Pseudomonas aeruginosa (PA). IRAK-M deletion, inflammation, myeloperoxidase (MPO) activity and PA load were measured in leukocytes, bronchoalveolar lavage (BAL) fluid and lungs. PA killing assay with BAL fluid was performed to determine mechanisms of IRAK-M-mediated host defense. IRAK-M mRNA and protein levels in alveolar and lung macrophages were significantly reduced in LysM–Cre+/IRAK-Mfl/wt mice compared with control mice. Following PA infection, LysM–Cre+/IRAK-Mfl/wt mice have enhanced lung neutrophilic inflammation, including MPO activity, but reduced PA load. The increased lung MPO activity in LysM–Cre+/IRAK-Mfl/wt mouse BAL fluid reduced PA load. Generation of IRAK-M conditional knockout mice will enable investigators to determine precisely the function of IRAK-M in myeloid cells and other types of cells during infection and inflammation.