The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Checkpoints in lymphocyte development and autoimmune disease.

Antigen receptor-controlled checkpoints in B lymphocyte development are crucial for the prevention of autoimmune diseases such as systemic lupus erythematosus. Checkpoints at the stage of pre-B cell receptor (pre-BCR) and BCR expression can eliminate certain autoreactive BCRs either by deletion of or anergy induction in cells expressing autoreactive BCRs or by receptor editing. For T cells, the picture is more complex because there are regulatory T (T(reg)) cells that mediate dominant tolerance, which differs from the recessive tolerance mediated by deletion and anergy. Negative selection of thymocytes may be as essential as T(reg) cell generation in preventing autoimmune diseases such as type 1 diabetes, but supporting evidence is scarce. Here we discuss several scenarios in which failures at developmental checkpoints result in autoimmunity.

ß-Selection-induced proliferation is required for αß T cell differentiation.

Proliferation and differentiation are tightly coordinated to produce an appropriate number of differentiated cells and often exhibit an antagonistic relationship. Developing T cells, which arise in the thymus from a minute number of bone-marrow-derived progenitors, undergo a major expansion upon pre-T cell receptor (TCR) expression. The burst of proliferation coincides with differentiation toward the αß T cell lineage-but the two processes were previously thought to be independent from one another, although both were driven by signaling from pre-TCR and Notch receptors. Here we report that proliferation at this step was not only absolutely required for differentiation but also that its ectopic activation was sufficient to substantially rescue differentiation in the absence of Notch signaling. Consistently, pharmacological inhibition of the cell cycle machinery also blocked differentiation in vivo. Thus the proliferation step is strictly required prior to differentiation of immature thymocytes.

Notch 1 keeps pro-T cells on track.

In this issue of Immunity, Feyerabend et al. (2009) report that Delta-like 4, acting on Notch 1, prevents pro-T cells from differentiating into dendritic cells and B cells. In addition, in the absence of Notch 1, B cells in the thymus arose from a cell-extrinsic pathway.

Unique features of the pre-T-cell receptor alpha-chain: not just a surrogate.

The pre-T-cell receptor (pre-TCR) has a crucial role in the normal development of alphabeta T cells. Different views have emerged concerning the structure and function of the pre-TCR. This molecular complex can be viewed as a variant of the alphabeta-TCR in which the pre-TCR alpha-chain that is covalently associated with the TCR beta-chain is a 'surrogate' TCR alpha-chain. Alternatively, the unique structure of the pre-TCR might be associated with a unique function, owing to evolutionary selection of a pre-TCR alpha-chain that has different capabilities from the TCR alpha-chain. As described here, I consider that experimental evidence favours the latter view.

Notch 1 activation in the molecular pathogenesis of T-cell acute lymphoblastic leukaemia.

The chromosomal translocation t(7;9) in human T-cell acute lymphoblastic leukaemia (T-ALL) results in deregulated expression of a truncated, activated form of Notch 1 (TAN1) under the control of the T-cell receptor-beta (TCRB) locus. Although TAN1 efficiently induces T-ALL in mouse models, t(7;9) is present in less than 1% of human T-ALL cases. The recent discovery of novel activating mutations in NOTCH1 in more than 50% of human T-ALL samples has made it clear that Notch 1 is far more important in human T-ALL pathogenesis than previously suspected.

Extra-thymically induced regulatory T cells: do they have potential in disease prevention?

Fopx3(+) Treg safeguard against autoimmune diseases and immune pathology. The extrathymic conversion of naïve T cells into Foxp3(+) regulatory T cells can be achieved in vivo by the delivery of strong-agonist ligands under subimmunogenic conditions. Tolerogenic vaccination with strong-agonist mimetopes of self-antigen to promote self-antigen specific tolerance may represent the most specific and safest means of preventing autoimmunity. This review discusses the requirements for induction of dominant tolerance exerted by Foxp3(+) Tregs in autoimmunity with special emphasis on their impact to interfere with T1D. The future goals are the understanding of self-non-self discrimination at the cellular and molecular level, which should then enable investigators to develop clinical vaccination protocols that specifically interfere with unwanted immune responses.

gammadeltaTCR ligands and lineage commitment.

Two major T lymphocyte lineages--alphabeta and gammadelta T cells--develop in the thymus from common precursors. Differentiation of both lineages requires signals coming from TCRs. Development of alphabeta T cells is driven at early stages by signaling from the pre-TCR, most likely in a ligand-independent fashion, and later--by signals delivered by alphabetaTCRs binding to their ligands--classical or non-classical MHC molecules. gammadelta lineage cells likewise require TCR signaling for their differentiation. Recent work from several groups suggests that TCR signaling not only ensures the developmental progression towards alphabeta and gammadelta lineages but that signal strength instructs lineage fate: weaker TCR signal results in alphabeta and stronger--in gammadelta lineage commitment. However, as most gammadeltaTCRs remain orphan receptors, it is still debated whether strong signals from gammadeltaTCRs in development are generated in a ligand-dependent manner (as in the case of alphabetaTCRs), ligand-independent manner (as for pre-TCR) or both. Here we summarize evidence supporting a possible role for ligands in gammadelta T cell lineage commitment and the generation of gammadelta sublineages.

αß versus γδ fate choice: counting the T-cell lineages at the branch point.

Both αß and γδ T cells develop in the thymus from a common progenitor. Historically distinguished by their T-cell receptor (TCR), these lineages are now defined on the basis of distinct molecular programs. Intriguingly, in many transgenic and knockout systems these programs are mismatched with the TCR type, leading to the development of γδ lineage cells driven by αßTCR and vice versa. These puzzling observations were recently explained by the demonstration that TCR signal strength, rather than TCR type per se, instructs lineage fate, with stronger TCR signal favoring γδ and weaker signal favoring αß lineage fates. These studies also highlighted the ERK (extracellular signal regulated kinase)-Egr (early growth response)-Id3 (inhibitor of differentiation 3) axis as a potential molecular switch downstream of TCR that determines lineage choice. Indeed, removal of Id3 was sufficient to redirect TCRγδ transgenic cells to the αß lineage, even in the presence of strong TCR signal. However, in TCR non-transgenic Id3 knockout mice the overall number of γδ lineage cells was increased due to an outgrowth of a Vγ1Vδ6.3 subset, suggesting that not all γδ T cells depend on this molecular switch for lineage commitment. Thus, the γδ lineage may in fact be a collection of two or more lineages not sharing a common molecular program and thus equipollent to the αß lineage. TCR signaling is not the only factor that is required for development of αß and γδ lineage cells; other pathways, such as signaling from Notch and CXCR4 receptors, cooperate with the TCR in this process.

Oral tolerance: is it all retinoic acid?

Oral tolerance has been argued to depend on "special" presentation of antigen in the gut. New studies support this idea by showing that the catalysis of vitamin A into retinoic acid (RA) in gut-associated dendritic cells (DCs) enhances the transforming growth factor (TGF)-beta-dependent conversion of naive T cells into regulatory T (T reg) cells and also directs T reg cell homing to the gut. These results reveal new tolerance mechanisms that will aid the use of T reg cells in the clinic.

Foxp3 occupancy and regulation of key target genes during T-cell stimulation.

Foxp3+CD4+CD25+ regulatory T (T(reg)) cells are essential for the prevention of autoimmunity. T(reg) cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in T(reg) cells, is required for T(reg) development and function, and is sufficient to induce a T(reg) phenotype in conventional CD4+CD25- T cells. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse. FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes. However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for T(reg) function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress the activation of target genes on T-cell stimulation. Foxp3 suppression of its targets appears to be crucial for the normal function of T(reg) cells, because overactive variants of some target genes are known to be associated with autoimmune disease.

Enhancement of antigen-specific Treg vaccination in vivo.

The conversion of naive T cells into Treg can be achieved in vivo by delivery of antigen under subimmunogenic conditions. Here we have examined several drugs for their ability to enhance the conversion process in vivo and have found that the rapamycin analog everolimus potently enhances Treg conversion by interfering with T-cell costimulation, reducing cell division and thereby activation of DNA methyltransferase 1 as well as by reducing T-cell activation through the ATP-gated P2×7 receptor controlling Ca2(+) influx. The resulting Tregs exhibit increased stability of Foxp3 expression even when generated in TGFß-containing media in vitro. Thus the mammalian target of rapamycin (mTOR) inhibitor everolimus in addition to inhibiting immune responses enhances Treg conversion by several distinct pathways. The converted Tregs can be further expanded by injection of IL-2/IL-2ab complexes. These complexes also increase the number of CD25(+)Foxp3(-) cells that, however, do not represent cytokine secreting effector cells but anergic cells, some of which can secrete IL-10 and can themselves be considered regulatory T cells as well. The combined use of everolimus and IL-2/IL-2ab complexes in vivo makes it feasible to achieve highly effective antigen-driven conversion of naive T cells into Treg and their expansion in vivo and thereby the described protocols constitute important tools to achieve immunological tolerance by Treg vaccination.

Genomic definition of multiple ex vivo regulatory T cell subphenotypes.

Regulatory T (Treg) cells that express the Foxp3 transcription factor are essential for lymphoid homeostasis and immune tolerance to self. Other nonimmunological functions of Treg cells, such as controlling metabolic function in adipose tissue, are also emerging. Treg cells originate primarily in the thymus, but can also be elicited from conventional T cells by in vivo exposure to low-dose antigen or homeostatic expansion or by activation in the presence of TGFbeta in vitro. Treg cells are characterized by a distinct transcriptional signature controlled in part, but not solely, by Foxp3. For a better perspective on transcriptional control in Treg cells, we compared gene expression profiles of a broad panel of Treg cells from various origins or anatomical locations. Treg cells generated by different means form different subphenotypes and were identifiable by particular combinations of transcripts, none of which fully encompassed the entire Treg signature. Molecules involved in Treg cell effector function, chemokine receptors, and the transcription factors that control them were differentially represented in these subphenotypes. Treg cells from the gut proved dissimilar to cells elicited by exposure to TGFbeta in vitro, but instead they resembled a CD103(+)Klrg1(+) subphenotype preferentially generated in response to lymphopenia.

Promoting tolerance to proteolipid protein-induced experimental autoimmune encephalomyelitis through targeting dendritic cells.

In T cell-mediated autoimmune diseases, self-reactive T cells with known antigen specificity appear to be particularly promising targets for antigen-specific induction of tolerance without compromising desired protective host immune responses. Several lines of evidence suggest that delivery of antigens to antigen-presenting dendritic cells (DCs) in the steady state (i.e., to immature DCs) may represent a suitable approach to induce antigen-specific T-cell tolerance peripherally. Here, we report that anti-DEC205-mediated delivery of the self-peptide proteolipid protein (PLP)139-151 to DCs ameliorated clinical symptoms in the PLP-induced SJL model of experimental autoimmune encephalomyelitis. Splenocytes from treated mice were anergized to PLP139-151, and IL-17 secretion was markedly reduced. Moreover, we show directly, using transgenic CD4(+) Vß6(+) TCR T cells specific for PLP139-151, that, under the conditions of the present experiments, these cells also became anergic. In addition, evidence for a CD4(+) T cell-mediated suppressor mechanism was obtained.

Phenotypic plasticity of T cell progenitors upon exposure to Notch ligands.

Despite many efforts, the nature of thymic immigrants that give rise to T cells has remained obscure, especially since it became known that extrathymic lineage-negative, Sca-1-positive, c-kit high progenitor cells differ from intrathymic early T cell progenitors (ETPs) by functional potential and dependence on Notch signaling. After our observation that intrathymic T cell precursors expressing a human CD25 reporter under control of pre-TCRalpha regulatory elements almost exclusively have the ETP phenotype, we have analyzed the phenotypic changes of reporter-expressing common lymphoid progenitor (CLP) cells in the bone marrow when cultured on Delta-like 1-expressing stromal cells. We note that these quickly adopt the phenotype of double negative (DN)2 thymocytes with little display of the ETP phenotype. Our data suggest that common lymphoid progenitor (CLP) cells could be responsible for the rapid reconstitution of thymus function after bone marrow transplantation since CLP cells in the blood have the capacity to rapidly enter the thymus and become DN2 thymocytes.

The E delta enhancer controls the generation of CD4- CD8- alphabetaTCR-expressing T cells that can give rise to different lineages of alphabeta T cells.

It is well established that the pre-T cell receptor for antigen (TCR) is responsible for efficient expansion and differentiation of thymocytes with productive TCRbeta rearrangements. However, Ptcra- as well as Tcra-targeting experiments have suggested that the early expression of Tcra in CD4- CD8- cells can partially rescue the development of alphabeta CD4+ CD8+ cells in Ptcra-deficient mice. In this study, we show that the TCR E delta but not E alpha enhancer function is required for the cell surface expression of alphabetaTCR on immature CD4- CD8- T cell precursors, which play a crucial role in promoting alphabeta T cell development in the absence of pre-TCR. Thus, alphabetaTCR expression by CD4- CD8- thymocytes not only represents a transgenic artifact but occurs under physiological conditions.

Differential synergy of Notch and T cell receptor signaling determines alphabeta versus gammadelta lineage fate.

Thymic precursors expressing the pre-T cell receptor (TCR), the gammadeltaTCR, or the alphabetaTCR can all enter the CD4+ 8+ alphabeta lineage, albeit with different efficacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into alphabeta lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still significantly contributes to the generation of alphabeta T cells. In particular, in alphabeta lineage commitment, the pre-TCR synergizes more efficiently with Notch signals than the other two TCRs, whereas gammadeltaTCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of alphabeta versus gammadelta lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the efficacy of alphabeta T cell generation.

Multiple extrathymic precursors contribute to T-cell development with different kinetics.

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches.