Γδ NKT Cells Research Articles

Single-cell proteogenomics reveals that SLAM/SAP signaling regulates the development of innate-like γδ T cell subsets with distinct TCR repertoires

Abstract γδ T cells are non-conventional T cells that are highly enriched in the mucosal tissues where they play critical roles in immunity. We recently demonstrated that the SLAM/SAP signaling pathway regulates the thymic development of innate-like γδT17 and γδTIFN subsets, in addition to γδNKT cells. Here, we utilized a single-cell proteogenomics approach coupled with γδ V(D)J profiling to define the transcriptional landscape and developmental checkpoints of SAP-dependent γδ T cells. This analysis not only confirmed our previous finding that SLAMF1 and SLAMF6 expression marks γδT17 and γδTIFN subsets, respectively, it also identified SLAMF7 as a novel marker of the SAP-dependent innate-like CD44+ CD45RB+ γδTIFN cells in both the thymus and periphery. Next, our data indicated that disruption of SAP-dependent signaling impaired γδT17 development at a very early (CD24high CD73−) stage, and was associated with the decreased expression of critical regulators of γδT17 development such as Blk and c-Maf. In contrast, while SAP also impaired γδTIFN development at an early (CD24high CD73+) stage, SAP-deficient γδTIFN cells exhibited increased expression of genes associated with TCR signaling and thymic export such as Prkch, Dgka, Klf2, and S1p1r. Finally, our analysis revealed significant alterations in the γδT17 TCR repertoire in both embryonic/neonatal thymus and the lung, as well as the presence of a SAP-dependent IFN-γ-producing lung Vγ4 population that preferentially utilized TRDV7. Altogether, these data suggest that SLAM/SAP signaling acts during the very early stages of γδ T cell development where it regulates critical pathways in both γδT17 and γδTIFN development, and influences the development of the innate-like γδ TCR repertoire. Supported by NIH (R03AI153902, P30GM118228), AAI Careers in Immunology Fellowship

Recognition of synthetic polyanionic ligands underlies "spontaneous" reactivity of Vγ1 γδTCRs.

Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1‐containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self‐ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate‐like αβ T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long‐standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation‐independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

Sonic Hedgehog Is a Determinant of γδ T-Cell Differentiation in the Thymus

Here we investigate the function of Hedgehog (Hh) signaling in thymic γδ T-cell maturation and subset differentiation. Analysis of Hh mutants showed that Hh signaling promotes γδ T-cell development in the thymus and influences γδ T-cell effector subset distribution. Hh-mediated transcription in thymic γδ cells increased γδ T-cell number, and promoted their maturation and increased the γδNKT subset, whereas inhibition of Hh-mediated transcription reduced the thymic γδ T-cell population and increased expression of many genes that are normally down-regulated during γδ T-cell maturation. These changes were also evident in spleen, where increased Hh signaling increased γδNKT cells, but reduced CD27-CD44+ and Vγ2+ populations. Systemic in vivo pharmacological Smoothened-inhibition reduced γδ T-cell and γδNKT cells in the thymus, and also reduced splenic γδ T-cell and γδNKT populations, indicating that Hh signaling also influences homeostasis of peripheral γδ T-cell populations. Taken together our data indicate that Sonic Hedgehog is an important determinant of γδ T-cell effector subset differentiation.

Derivation of mimetic γδ T cells endowed with cancer recognition receptors from reprogrammed γδ T cell.

Using induced pluripotent stem cells (iPSCs) to derive chimeric antigen receptor-modified T (CAR-T) cells has great industrial potential. A previous study used αβ T cell-derived CAR-modified iPSCs to produce CAR-T cells. However, these αβ T cells are restricted to autologous use and only recognize single cancer antigen. To make CAR-T alternative for allogeneic use, we reprogrammed γδ T cell into iPSCs (γδ T-iPSCs) to circumvent the risk of graft-versus-host disease. To target multiple cancer-associated antigens, we used an “NK cell-promoting” protocol to differentiate γδ T-iPSCs and to induce expression of natural killer receptors (NKRs). Through such two-step strategy, mimetic γδ T cells endowed with an array of NKRs and thus designated as “γδ natural killer T (γδ NKT) cells” were derived. With no/low-level expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) and immune checkpoint receptors, γδ NKT cells may provide a potent “off-the-shelf” cytotoxic cell source to recognize multiple ubiquitous antigens in a broad spectrum of cancers.

Styk1 is specifically expressed in NK1.1+ lymphocytes including NK, γδ T, and iNKT cells in mice, but is dispensable for their ontogeny and function.

Innate Tcells, NK cells, and innate-like lymphocytes (ILCs) share transcriptional signatures that translate into overlapping developmental and functional programs. A prominent example for genes that are highly expressed in NK cells but not in ILCs is serine-threonine-tyrosine kinase 1 (Styk1 encoded by Styk1). We found Styk1 to be specifically expressed in lymphocytes positive for Killer cell lectin-like receptor subfamily B, member 1, also known as CD161 or NK1.1, i.e. in NK cell, αβ iNKT, and γδ NKTcell lineages. To investigate the role of Styk1 in the development and function of NK1.1+ innate T-cell subsets, we generated and analyzed a novel Styk1null mutant mouse line. Furthermore, we validated Styk1 expression in γδ NKTcells and in thymic, but not in peripheral invariant αβ iNKTcells through ex vivo analysis of a concomitantly generated transgenic Styk1 reporter mouse line. Despite the very specific expression of Styk1 in NK cells, γδ NKTcells, and thymic αβ iNKT, its absence did not alter homeostasis and function of these lineages. Thus, Styk1 expression is specific for NK cells and selected NK-like innate T-cell subsets, but dispensable for their development and function.

Id3 Restricts γδ NKT Cell Expansion by Controlling Egr2 and c-Myc Activity.

γδ NKT cells are neonatal-derived γδ T lymphocytes that are grouped together with invariant NKT cells based on their shared innate-like developmental program characterized by the transcription factor PLZF (promyelocytic leukemia zinc finger). Previous studies have demonstrated that the population size of γδ NKT cells is tightly controlled by Id3-mediated inhibition of E-protein activity in mice. However, how E proteins promote γδ NKT cell development and expansion remains to be determined. In this study, we report that the transcription factor Egr2, which also activates PLZF expression in invariant NKT cells, is essential for regulating γδ NKT cell expansion. We observed a higher expression of Egr family genes in γδ NKT cells compared with the conventional γδ T cell population. Loss of function of Id3 caused an expansion of γδ NKT cells, which is accompanied by further upregulation of Egr family genes as well as PLZF. Deletion of Egr2 in Id3-deficient γδ NKT cells prevented cell expansion and blocked PLZF upregulation. We further show that this Egr2-mediated γδ NKT cell expansion is dependent on c-Myc. c-Myc knockdown attenuated the proliferation of Id3-deficient γδ NKT cells, whereas c-Myc overexpression enhanced the proliferation of Id3/Egr2-double-deficient γδ NKT cells. Therefore, our data reveal a regulatory circuit involving Egr2-Id3-E2A, which normally restricts the population size of γδ NKT cells by adjusting Egr2 dosage and c-Myc expression.

Three distinct developmental pathways for adaptive and two IFN-\u03b3-producing \u03b3\u03b4 T subsets in adult thymus

Murine γδ T cells include subsets that are programmed for distinct effector functions during their development in the thymus. Under pathological conditions, different γδ T cell subsets can be protective or can exacerbate a disease. Here we show that CD117, CD200 and CD371, together with other markers, identify seven developmental stages of γδ T cells. These seven stages can be divided into three distinct developmental pathways that are enriched for different TCRδ repertoires and exhibit characteristic expression patterns associated with adaptive (γδTn), IFN-γ-producing (γδT1) and IFN-γ/IL-4-co-producing γδ T cells (γδNKT). Developmental progression towards both IFN-γ-producing subsets can be induced by TCR signalling, and each pathway results in thymic emigration at a different stage. Finally, we show that γδT1 cells are the predominating IFN-γ-producing subset developing in the adult thymus. Thus, this study maps out three distinct development pathways that result in the programming of γδTn, γδT1 and γδNKT cells.

MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development.

Thymic development of αβ T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1–/–mice, which show impaired agonistic T cell selection of invariant αβ NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αβ NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes.

PLZF Controls the Development of Fetal-Derived IL-17+Vγ6+ γδ T Cells.

Expression of promyelocytic leukemia zinc finger (PLZF) protein directs the effector differentiation of invariant NKT (iNKT) cells and IL-4(+) γδ NKT cells. In this study, we show that PLZF is also required for the development and function of IL-17(+) γδ T cells. We observed that PLZF is expressed in fetal-derived invariant Vγ5(+) and Vγ6(+) γδ T cells, which secrete IFN-γ and IL-17, respectively. PLZF deficiency specifically affected the effector differentiation of Vγ6(+) cells, leading to reduced numbers of mature CD27(-)CD44(+) phenotype capable of secreting IL-17. Although PLZF was not required for Vγ5(+) γδ T cells to develop, when these cells were reprogrammed into IL-17-secreting cells in Skint-1 mutant mice, they required PLZF for their effector maturation, similarly to Vγ6(+) γδ T cells. The impaired effector differentiation of PLZF-deficient Vγ6(+) γδ T cells was not due to increased apoptosis and it was related to reduced proliferation of immature CD27(+)CD44(-) Vγ6(+) γδ T cells, which was required for their differentiation into mature CD27(-)CD44(+) IL-17-secreting cells. Thus, the present study identifies that PLZF function is not restricted to NKT or IL-4(+) T cells, but it also controls the development of IL-17(+) γδ T cells.

Lymphocytes in atherosclerosis.

Atherosclerosis is the major cause of cardiovascular disease, and cardiovascular disease remains the primary cause of death in the United States. The primary complication for patients with type 2 diabetes mellitus (T2D) is cardiovascular disease.1–8 Because the incidences of T2D and obesity are drastically increasing in the United States and now worldwide, understanding how atherosclerosis is accelerated in these diseases becomes even more important to discern. Although many scientific advances in research on atherosclerosis have been made by multiple groups during the past 30 years, particularly about the function of endothelial and smooth muscle biology and macrophage foam cell formation in atherosclerotic plaque development, the roles of immune cells in regulating atherosclerosis onset and progression are still being elucidated. It was widely thought that immune cells played little part in atherogenesis until 1986, when Hansson et al9–12 reported the presence of lymphocytes within atherosclerotic lesions; yet it was many years later until it was widely accepted that immune cells play an important role in atherogenesis. Since 1986, the viewpoint has changed significantly, but details on lymphocyte subsets and mechanisms are still emerging. Moreover, controversy still exists on the roles that some lymphocyte subsets play in atherosclerosis. These discrepancies in findings are at least partly because of differences in mouse models used, diet composition, and length of time on diet for atherosclerosis measurements. However, despite a few ongoing controversies, there are many studies on immune cells in atherogenesis in mice that show clear findings.10,12–23 For example, CD4+ T lymphocytes have been shown to accelerate atherosclerosis.18,24–29 Activated CD4 T cells can be distinguished based on their phenotype, and the predominant CD4+ effector T cells studied to date in the context of atherosclerosis are Th1, …

CD28 controls the development of innate‐like CD8+T cells by promoting the functional maturation of NKT cells

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αβ NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger(+) IL-4(+) NKT cells and upregulation of LFA-1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28-deficient mice is cell autonomous. Moreover, we show in both wild-type C57BL/6 mice and in downstream of tyrosine kinase-1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28-mediated regulation of thymic IL-4(+) NKT cells promotes the differentiation of eomesodermin(+) CD44(high) innate-like CD8(+) T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT-cell homeostasis and the size of the innate-like CD8(+) T-cell pool.

The Tec Kinase ITK Regulates Thymic Expansion, Emigration, and Maturation of γδ NKT Cells

The Tec family tyrosine kinase, Itk, regulates signaling downstream of the TCR. The absence of Itk in CD4(+) T cells results in impaired Th2 responses along with defects in maturation, cytokine production, and survival of iNKT cells. Paradoxically, Itk(-/-) mice have spontaneously elevated serum IgE levels, resulting from an expansion of the Vγ1.1(+)Vδ6.3(+) subset of γδ T cells, known as γδ NKT cells. Comparisons between γδ NKT cells and αβ iNKT cells showed convergence in the pattern of cell surface marker expression, cytokine profiles, and gene expression, suggesting that these two subsets of NKT cells undergo similar differentiation programs. Hepatic γδ NKT cells have an invariant TCR and are derived predominantly from fetal progenitors that expand in the thymus during the first weeks of life. The adult thymus contains these invariant γδ NKT cells plus a heterogeneous population of Vγ1.1(+)Vδ6.3(+) T cells with diverse CDR3 sequences. This latter population, normally excluded from the liver, escapes the thymus and homes to the liver when Itk is absent. In addition, Itk(-/-) γδ NKT cells persistently express high levels of Zbtb16 (PLZF) and Il4, genes that are normally downregulated in the most mature subsets of NKT cells. These data indicate that Itk signaling is required to prevent the expansion of γδ NKT cells in the adult thymus, to block their emigration, and to promote terminal NKT cell maturation.

Induced IL-17–Producing Invariant NKT Cells Require Activation in Presence of TGF-β and IL-1β

IL-17 production by innate-like lymphocytes, including γδ and invariant NKT (iNKT) cells, have been ascribed to specific lineages that are endowed with this functional specialization during thymic differentiation. IL-17-producing iNKT cells have been described as a CD4(-)NK1.1(-) lineage in mice and CD161(+) in humans. We found that, in mice, noncommitted iNKT cells can be induced to produce IL-17 when activated in presence of TGF-β and IL-1β. This peripheral induction of IL-17 expression could be observed in any subset irrespectively of CD4 and NK1.1 expression, the process leading to loss of NK1.1 expression and partial CD4 downmodulation. Furthermore, induced IL-17-producing iNKT cells were sufficient to drive neutrophilic airways inflammation upon intratracheal adoptive cell transfer into congenic mice. Taken together, our data show that similarly to regulatory T cells, which have a natural and peripherally induced subset, IL-17 production by iNKT cells can also be imprinted in natural iNKT17 cells or peripherally induced.

Development of PLZF-expressing γδ NKT cells in Itk-/- mice (115.13)

Abstract In conventional αβ T cells, the Tec family tyrosine kinase, Itk is required for signaling downstream of the TCR. Itk also regulates αβ T cell development, lineage commitment, and effector function. A well-established feature of Itk-/- mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE. We have previously shown the cause of this phenotype is due to the increase of the mature Vγ1.1+Vδ6.3+ (V6) subset of γδ T cells in Itk-/- mice. Wild type V6 γδ T cells display characteristics similar to αβ NKT cells including dual secretion of IL-4 and IFNγ along with expression of the transcription factor PLZF; therefore they are γδ NKT cells. Along with increased proportions of mature V6 cells in Itk-/- mice, V6 cells in Itk-/- mice have increased IL-4 secretion and elevated levels of PLZF expression. Based upon PLZF expression and the use of the IL-4 reporter (4Get) mice, our data indicate that γδ NKT cells have similar differentiation stages as αβ NKT cells, and that Itk-/- γδ NKT cells are blocked during development. Together, these data indicate that Itk signaling regulates γδ T cell lineage development and effector function, and is required to control IgE production in vivo.

Endogenous mouse mammary tumor viruses influence the development of thymic αβ NKT cells and innate CD8+ T cells (111.26)

Abstract Mounting evidence has demonstrated the presence of a population of thymic CD8+ T cells that share phenotypic and functional similarity to “innate-like” or “memory-like” T cells. Innate CD8+ T cells have increased expression of CD44, CD122, and Eomesodermin (Eomes), as well as the capacity to rapidly produce interferon (IFN)-γ upon stimulation. In addition to several gene-deficient models, including KLF2-, Itk-, and Id3-deficient mice, innate CD8+ T cells have also been documented in naïve BALB/c mice. The development of such cells is dependent on the production of interleukin (IL)-4 from an expanded population of αβ or γδ NKT cells expressing the transcription factor promyelocytic leukemia zinc finger (PLZF). Interestingly, this innate phenotype among CD8+ T cells is lost in congenic BALB/c mice lacking all endogenous mouse mammary tumor proviruses (BALB/Mtv-null). In the absence of endogenous Mtv proviruses, the expression of CD44, CD122, CXCR3, and Eomes, as well as the production of IFN-γ, was greatly reduced among thymic CD8+ T cells. The loss of this innate CD8+ T cell population in BALB/Mtv-null mice correlated with a reduction in PLZF+, IL-4 secreting αβ NKT cells within the thymus compared to wild type BALB/c. Our findings suggest a potential Mtv-mediated role in modulation of thymic αβ NKT cell development that subsequently influences the generation of innate CD8+ T cells.

Deletion of factors in the Id3 pathway results in a dramatic phenotype where Vγ1Vδ6.3 (γδ NKT) T cells become the dominant T lymphocyte (113.2)

Abstract Recent work from our lab and others has shown a significant expansion of γδ NKT cells in mice that lack the HLH-protein Id3. In contrast to WT mice, nearly all Id3-deficient γδ NKT cells express high levels of the innate T cell determinant, PLZF and produce copious amounts of IL-4 upon activation. We have been exploring the transcriptional network that controls the development of γδ NKT cells. In an effort to identify additional factors that play a key role in expansion of γδ NKT cells, we have undertaken a candidate approach to evaluate genes that function in the Id3 pathway. To do this we are studying single and double gene deficient mice to determine the interrelation of different transcription factors. These studies have led to the discovery of a gene combination that results in a dramatic alteration of thymopoiesis. In these mice, while thymic cellularity was not affected, the frequency of γδ T cells versus αβ T cells was reversed. γδ T cells were found to be the dominant lymphocyte, representing ~65% of the cells. Furthermore, nearly all of the γδ T cells were now found to express PLZF. This dramatic phenotype appears to be T cell-specific, as both myeloid and lymphoid lineages are present. Analysis of thymic progenitors revealed major alterations in DN2-DN4 stages of development. Our findings uncover a fundamental regulatory pathway that favors the development of γδ NKT cells, while restricting the maturation of pro-T cells into the conventional T cell lineage.

Tec kinase Itk in γδT cells is pivotal for controlling IgE production in vivo

In conventional alphabeta T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the T cell receptor (TCR). Itk also regulates alphabeta T cell development, lineage commitment, and effector function. A well established feature of Itk(-/-) mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE and increased numbers of germinal center B cells. Here we show that the source of this phenotype is gammadelta T cells, as normal IgE levels are observed in Itk(-/-)Tcrd(-/-) mice. When stimulated through the gammadelta TCR, Itk(-/-) gammadelta T cells produce high levels of Th2 cytokines, but diminished IFNgamma. In addition, activated Itk(-/-) gammadelta T cells up-regulate costimulatory molecules important for B cell help, suggesting that they may directly promote B cell activation and Ig class switching. Furthermore, we find that gammadelta T cells numbers are increased in Itk(-/-) mice, most notably the Vgamma1.1(+)Vdelta6.3(+) subset that represents the dominant population of gammadelta NKT cells. Itk(-/-) gammadelta NKT cells also have increased expression of PLZF, a transcription factor required for alphabeta NKT cells, indicating a common molecular program between alphabeta and gammadelta NKT cell lineages. Together, these data indicate that Itk signaling regulates gammadelta T cell lineage development and effector function and is required to control IgE production in vivo.