A Distinctive γδ T Cell Repertoire in NOD Mice Weakens Immune Regulation and Favors Diabetic Disease.

Previous studies in mice and humans suggesting that γδ T cells play a role in the development of type 1 diabetes have been inconsistent and contradictory. We attempted to resolve this for the type 1 diabetes-prone NOD mice by characterizing their γδ T cell populations, and by investigating the functional contributions of particular γδ T cells subsets, using Vγ-gene targeted NOD mice. We found evidence that NOD Vγ4+ γδ T cells inhibit the development of diabetes, and that the process by which they do so involves IL-17 production and/or promotion of regulatory CD4+ αß T cells (Tregs) in the pancreatic lymph nodes. In contrast, the NOD Vγ1+ cells promote diabetes development. Enhanced Vγ1+ cell numbers in NOD mice, in particular those biased to produce IFNγ, appear to favor diabetic disease. Within NOD mice deficient in particular γδ T cell subsets, we noted that changes in the abundance of non-targeted T cell types also occurred, which varied depending upon the γδ T cells that were missing. Our results indicate that while certain γδ T cell subsets inhibit the development of spontaneous type 1 diabetes, others exacerbate it, and they may do so via mechanisms that include altering the levels of other T cells.

Becoming aware of γδ T cells.

The discovery that B cells and αß T cells exist was predictable: These cells gave themselves away through their products and biological effects. In contrast, there was no reason to anticipate the existence of γδ T cells. Even the accidental discovery of a novel TCR-like gene (later named γ) that did not encode TCR α or ß proteins did not immediately change this. TCR-like γ had no obvious function, and its early expression in the thymus encouraged speculation about a possible role in αß T cell development. However, the identification of human PBL-derived cell-lines which expressed CD3 in complex with the TCR-like γ protein, but not the αß TCR, first indicated that a second T cell-type might exist, and the TCR-like γ chain was observed to co-precipitate with another protein. Amid speculation about a possible second TCR, this potential dimeric partner was named δ. To determine if the δ protein was indeed TCR-like, we undertook to sequence it. Meanwhile, a fourth TCR-like gene was discovered and provisionally named x. TCR-like x had revealed itself through genomic rearrangements early in T cell development, and was an attractive candidate for the gene encoding δ. The observation that δ protein sequences matched the predicted amino acid sequences encoded by the x gene, as well as serological cross-reactivity, confirmed that the TCR-like x gene indeed encoded the δ protein. Thus, the γδ heterodimer was established as a second TCR, and the cells that express it (the γδ T cells) consequently represented a third lymphocyte-population with the potential of recognizing diverse antigens. Soon, it became clear that γδ T cells are widely distributed and conserved among the vertebrate species, implying biological importance. Consistently, early functional studies revealed their roles in host resistance to pathogens, tissue repair, immune regulation, metabolism, organ physiology and more. Albeit discovered late, γδ T cells have repeatedly proven to play a distinct and often critical immunological role, and now generate much interest.

Two functionally distinct subsets of IL-17 producing γδ T cells.

The γδ T cells play an important role in both mice and humans as a source of the cytokine IL-17, which is key for immune resistance to certain pathogens. In mice, most of these IL-17 producers, termed γδT-17 cells, actually comprise two distinct types: those expressing an invariant Vγ6Vδ1+ TCR and those expressing a Vγ4+ TCR. Murine γδT-17 cells acquire an inherent bias to produce IL-17 and other "type 17" cytokines during thymic development. The similarities and differences between the two mouse γδT-17 types are reviewed here, and the potential implications of their differences are discussed. There is some evidence that two distinct TCR-defined IL-17-producing γδ T cell subsets also exist in humans, but unlike the mouse γδT-17 cells, these cells are probably not imprinted with an IL-17 bias during thymic development, but rather acquire an IL-17 bias in the periphery.

BW5147 and Derivatives for the Study of T Cells and their Antigen Receptors.

Like B cells, T cells can be immortalized through hybridization with lymphoma cells, a technique that has been particularly useful in the study of the T cell receptors (TCR) for antigen. In T cell hybridizations, the AKR mouse strain-derived thymus lymphoma BW5147 is by far the most popular fusion line. However, the full potential of this technology had to await inactivation of the productively rearranged TCR-α and -ß genes in the lymphoma. BWα-ß-, the TCR-gene deficient variant of the original lymphoma, which has become the fusion line of choice for αß T cells, is now available with numerous modifications, enabling the investigation of many aspects of TCR-mediated responses and TCR-structure. Unexpectedly, inactivating BW's functional TCR-α gene also rendered the lymphoma more permissive for the expression of TCR-γδ, facilitating the study of γδ T cells, their TCRs, and their TCR-mediated reactivity.

γδ T cells shape memory-phenotype αß T cell populations in non-immunized mice.

Size and composition of γδ T cell populations change dramatically with tissue location, during development, and in disease. Given the functional differentiation of γδ T cell subsets, such shifts might alter the impact of γδ T cells on the immune system. To test this concept, and to determine if γδ T cells can affect other immune cells prior to an immune response, we examined non-immunized mice derived from strains with different genetically induced deficiencies in γδ T cells, for secondary changes in their immune system. We previously saw extensive changes in pre-immune antibodies and B cell populations. Here, we report effects on αß T cells. Similarly to the B cells, αß T cells evidently experience the influence of γδ T cells at late stages of their pre-immune differentiation, as single-positive heat stable antigen-low thymocytes. Changes in these and in mature αß T cells were most prominent with memory-phenotype cells, including both CD8+ and CD4+ populations. As previously observed with B cells, most of the effects on αß T cells were dependent on IL-4. Unexpectedly, IL-4 seemed to be produced mainly by αß T cells in the non-immunized mice, albeit strongly regulated by γδ T cells. Similarly to our findings with B cells, changes of αß T cells were less pronounced in mice lacking all γδ T cells than in mice lacking only some, suggesting that the composition of the γδ T cell population determines the nature of the γδ-influence on the other pre-immune lymphocytes.

Clonal Vγ6+Vδ4+ T cells promote IL-17-mediated immunity against Staphylococcus aureus skin infection.

T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL-17 response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1ß, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4 However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6+ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6+Vδ4+ T cells in immunity against S. aureus skin infections.

Correction: High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU.

[This corrects the article DOI: 10.1371/journal.pone.0199601.].

Connection between γδ T-cell- and Adenosine- Mediated Immune Regulation in the Pathogenesis of Experimental Autoimmune Uveitis.

Regulatory effects of γδ T-cells on immune responses have been studied for years. We have investigated the regulatory effect of γδ T-cells on Th1 and Th17 autoimmune responses, and have studied molecular and cellular mechanisms by which γδ T-cells enhance or inhibit immune responses, exploiting a well-characterized murine model of experimental autoimmune uveitis (EAU). Our results show that (1) aberrant γδ T-cell activation is an important pathogenic event in EAU; (2) γδ T-cells have a unique regulatory effect on Th17 autoimmune responses, which is shaped by the activation status of γδ T-cells; and (3) γδ-mediated immunoregulation is closely linked with the extracellular adenosine metabolism. Reciprocal interactions between γδ T-cells and extracellular adenosine partially determine the development of EAU.

Characterization of Mouse γδ T Cell Subsets in the Setting of Type-2 Immunity.

Accumulating evidence indicates that γδ T cells are a critical component of type-2 immunity. However, the role of these cells in type-2 immune responses seems to be divergent. γδ T cells are heterogeneous lymphocytes that can be further divided into TCR-Vγ/δ definable subsets. Different subsets have distinct and sometimes opposite function during immune responses. In this chapter, we describe the detailed protocol for characterization of γδ T cell subsets in a mouse model of ovalbumin (OVA)/alum-induced type-2 immunity. Our protocol includes identifying γδ T cell subsets by flow cytometry, functionally inactivating individual subsets in vivo, purifying γδ T cell subsets, and using adoptive cell transfer to explore the role of individual subsets in OVA/alum-induced IgE responses.

High level expression of A2ARs is required for the enhancing function, but not for the inhibiting function, of γδ T cells in the autoimmune responses of EAU.

We previously reported that activated γδ T cells greatly enhance autoimmune responses, particularly the Th17 response. To determine the mechanisms involved, we made a series of comparisons between activated and non-activated γδ T cells. Our results showed that activated γδ T cells expressed greatly increased levels of A2A adenosine receptor (A2AR) and decreased amounts of CD73, as well as increased amounts of T cell activation markers such as CD69, CD44 and CD25. We show that A2AR is a major functional molecule in the enhancing activity of γδ T cells. A2AR-/- γδ T cells (isolated from A2AR-/- mouse), lost their Th17-enhancing activity as did A2AR+/+ γδ T cells (isolated from wt-B6 mouse) after treatment with an A2AR antagonist. Since γδ T cells possess either an enhancing or an inhibiting effect, we also tested whether A2AR expression on γδ T cells is essential to their inhibiting effect. Our results showed that the inhibiting effect of A2AR-/- γδ T cells was as potent as that of A2AR+/+ γδ T cells. In a previous report we showed that the expression of different levels of CD73 molecule allowed γδ T cells to adjust their suppressive activity; in the current study, we show that expression of increased amounts of A2AR allows γδ T cells to more effectively exert their enhancing function.

Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

Nuclear membrane-localised NOX4D generates pro-survival ROS in FLT3-ITD-expressing AML.

Internal tandem duplication of the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3-ITD) is the most prevalent genetic aberration present in 20-30% of acute myeloid leukaemia (AML) cases and is associated with a poor prognosis. FLT3-ITD expressing cells express elevated levels of NADPH oxidase 4 (NOX4)-generated pro-survival hydrogen peroxide (H2O2) contributing to increased levels of DNA oxidation and double strand breaks. NOX4 is constitutively active and has been found to have various isoforms expressed at multiple locations within a cell. The purpose of this study was to investigate the expression, localisation and regulation of NOX4 28 kDa splice variant, NOX4D. NOX4D has previously been shown to localise to the nucleus and nucleolus in various cell types and is implicated in the generation of reactive oxygen species (ROS) and DNA damage. Here, we demonstrate that FLT3-ITD expressing-AML patient samples as well as -cell lines express the NOX4D isoform resulting in elevated H2O2 levels compared to FLT3-WT expressing cells, as quantified by flow cytometry. Cell fractionation indicated that NOX4D is nuclear membrane-localised in FLT3-ITD expressing cells. Treatment of MV4-11 cells with receptor trafficking inhibitors, tunicamycin and brefeldin A, resulted in deglycosylation of NOX4 and NOX4D. Inhibition of the FLT3 receptor revealed that the FLT3-ITD oncogene is responsible for the production of NOX4D-generated H2O2 in AML. We found that inhibition of the PI3K/AKT and STAT5 pathways resulted in down-regulation of NOX4D-generated pro-survival ROS. Taken together these findings indicate that nuclear membrane-localised NOX4D-generated pro-survival H2O2 may be contributing to genetic instability in FLT3-ITD expressing AML.

γδ T Cells and B Cells.

γδ T cells constitute the third arm of a tripartite adaptive immune system in jawed vertebrates, besides αß T cells and B cells. Like the other two lymphocyte-types, they express diverse antigen receptors, capable of specific ligand recognition. Functionally, γδ T cells represent a system of differentiated subsets, sometimes engaged in cross-regulation, which ultimately determines their effect on other components of the immune system, including B cells and antibodies. γδ T cells are capable of providing help to B cells in antibody production. More recently it became clear that γδ T cells influence B cell differentiation during the peripheral stages of B cell development, control levels of circulating immunoglobulin (all subclasses), and affect production of autoantibodies. Because of this relationship between γδ T cells and B cells, the extensive variation of γδ T cells among human individuals might be expected to modulate their humoral responsiveness.

Correction: CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

[This corrects the article DOI: 10.1371/journal.pone.0150078.].

Creativity in gifted identification: increasing accuracy and diversity.

Many federal definitions and popular theories of giftedness specify creativity as a core component. Nevertheless, states rely primarily on measures of intelligence for giftedness identification. As minority and culturally diverse students continue to be underrepresented in gifted programs, it is reasonable to ask if increasing the prominence of creativity in gifted identification may help increase balance and equity. In this paper, we explore both layperson and psychometric conceptions of bias and suggest that adding creativity measures to the identification process alleviates both perceptions and the presence of bias. We recognize, however, the logistic and measurement-related challenges to including creativity assessments.

A Special Connection between γδ T Cells and Natural Antibodies?

Natural antibodies (NAbs) play an important role in early host defense, autophagy and tissue remodeling, and in immune regulation. They arise spontaneously (without specific immunization), and are already present at birth. NAbs are produced by B1 B cells, MZ B cells and other B cell types. They include all major Ig subclasses but IgM antibodies are prevalent, especially early in development. NAbs may be poly-specific, recognize particular auto-antigens, or detect neo-determinants such as those exposed during apoptosis or generated by oxidation. NAbs do not require cognate T cell help but depend on soluble mediators produced by T cells. Our recent studies suggest that γδ T cells may have a special relationship with NAbs, and play a prominent role in their regulation, in part through the fine-tuning of IL-4 levels. The spontaneously activated state of these cells likely enables their cytokine production and other functions in the absence of external stimulation. Ontogenetically, the earlier arising γδ T cells are better positioned than αß T cells to shape the developing repertoire of NAbs. Intriguingly, ligand specificities of NAbs and γδ T cell receptors appear to be overlapping, perhaps allowing γδ cognate help for certain NAb specificities. Via NAbs, γδ T cells could exert a regulatory influence on numerous processes in health and disease.

CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

γδ T cells can either enhance or inhibit an adaptive immune response, but the mechanisms involved are not fully understood. Given that CD73 is the main enzyme responsible for conversion of AMP into the immunosuppressive molecule adenosine, we investigated its role in the regulatory function of γδ T cells in experimental autoimmune uveitis (EAU). We found that γδ T cells expressed different amounts of CD73 during the different stages of EAU and that low CD73 expression on γδ T cells correlated with enhanced Th17 response-promoting activity. Functional comparison of CD73-deficient and wild-type B6 (CD73+/+) mice showed that failure to express CD73 decreased both the enhancing and suppressive effects of γδ T cells on EAU. We also demonstrated that γδ T cells expressed different amounts of CD73 when activated by different pathways, which enabled them to either enhance or inhibit an adaptive immune response. Our results demonstrate that targeting CD73 expression on γδ T cells may allow us to manipulate their pro- or anti-inflammatory effect on Th17 responses.

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.