How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function.

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.

T cell-B cell thymic cross-talk: maintenance and function of thymic B cells requires cognate CD40-CD40 ligand interaction.

Thymic development requires bidirectional interaction or cross-talk between developing T cells and thymic stromal cells, a relationship that has been best characterized for the interaction between thymocytes and thymic epithelial cells. We have characterized in this article the requirement for similar cross-talk in the maintenance and function of thymic B cells, another population that plays a role in selection of developing thymic T cells. We found that maintenance of thymic B cells is strongly dependent on the presence of mature single-positive thymocytes and on the interactions of these T cells with specific Ag ligand. Maintenance of thymic B cell number is strongly dependent on B cell-autonomous expression of CD40, but not MHC class II, indicating that direct engagement of CD40 on thymic B cells is necessary to support their maintenance and proliferation. Thymic B cells can mediate negative selection of superantigen-specific, self-reactive, single-positive thymocytes, and we show that CD40 expression on B cells is critical for this negative selection. Cross-talk with thymic T cells is thus required to support the thymic B cell population through a pathway that requires cell-autonomous expression of CD40, and that reciprocally functions in negative selection of autoreactive T cells.

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

A novel T cell subset with trans-rearranged Vγ-Cß TCRs shows Vß expression is dispensable for lineage choice and MHC restriction.

αß T cells, which express the α-ß TCR heterodimer, express CD4 or CD8 coreceptors on cells that are MHC class I or MHC class II dependent. In contrast, γδ T cells do not express CD4 or CD8 and develop independently of MHC interaction. The factors that determine αß and γδ lineage choice are not fully understood, and the determinants of MHC restriction of TCR specificity have been controversial. In this study we have identified a naturally occurring population of T cells expressing Vγ-Cß receptor chains on the cell surface, the products of genomic trans-rearrangement between the Vγ2 gene and a variety of Dß or Jß genes, in place of an intact TCRß-chain and in association with TCRα. Identification of this population allowed an analysis of the role of TCR variable regions in determining T cell lineage choice and MHC restriction. We found that Vγ2(+)Cß(+) cells are positive for either CD4 or CD8 and are selected in an MHC class II- or MHC class I-dependent manner, respectively, thus following the differentiation pathway of αß and not γδ cells and demonstrating that Vß V region sequences are not required for selection of an MHC-restricted repertoire.

Thymic medullary epithelium and thymocyte self-tolerance require cooperation between CD28-CD80/86 and CD40-CD40L costimulatory pathways.

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTß, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.

TRAF3 enforces the requirement for T cell cross-talk in thymic medullary epithelial development.

Induction of self-tolerance in developing T cells depends on medullary thymic epithelial cells (mTECs), whose development, in turn, requires signals from single-positive (SP) thymocytes. Thus, the absence of SP thymocytes in Tcra(-/-) mice results in a profound deficiency in mTECs. Here, we have probed the mechanism that underlies this requirement for cross-talk with thymocytes in medullary development. Previous studies have implicated nonclassical NF-κB as a pathway important in the development of mTECs, because mice lacking RelB, NIK, or IKKα, critical components of this pathway, have an almost complete absence of mTECs, with resulting autoimmune pathology. We therefore assessed the effect of selective deletion in TEC of TNF receptor-associated factor 3 (TRAF3), an inhibitor of nonclassical NF-κB signaling. Deletion of TRAF3 in thymic epithelial cells allowed RelB-dependent development of normal numbers of AIRE-expressing mTECs in the complete absence of SP thymocytes. Thus, mTEC development can occur in the absence of cross-talk with SP thymocytes, and signals provided by SP T cells are needed to overcome TRAF3-imposed arrest in mTEC development mediated by inhibition of nonclassical NF-κB. We further observed that TRAF3 deletion is also capable of overcoming all requirements for LTßR and CD40, which are otherwise necessary for mTEC development, but is not sufficient to overcome the requirement for RANKL, indicating a role for RANKL that is distinct from the signals provided by SP thymocytes. We conclude that TRAF3 plays a central role in regulation of mTEC development by imposing requirements for SP T cells and costimulation-mediated cross-talk in generation of the medullary compartment.

Lck availability during thymic selection determines the recognition specificity of the T cell repertoire.

Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αß T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αßTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αßTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αßTCR repertoire.

Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells.

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4(+) T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4(+) T cells.

Upregulation of CD4 expression during MHC class II-specific positive selection is essential for error-free lineage choice.

The lineage fate of developing thymocytes is determined by the persistence or cessation of T cell receptor (TCR) signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We show here that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during major histocompatibility complex class II (MHC II)-specific positive selection and is critical for error-free lineage choice in TCR-transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurred for endogenously encoded CD4 coreceptors, but CD4 transgenes were downregulated during positive selection, disrupting MHC II-specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC II-specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice.

Deletion of CD4 and CD8 coreceptors permits generation of alphabetaT cells that recognize antigens independently of the MHC.

The thymus generates major histocompatibility complex (MHC)-restricted alphabetaT cells that only recognize antigenic ligands in association with MHC or MHC-like molecules. We hypothesized that MHC specificity might be imposed on a broader alphabetaTCR repertoire during thymic selection by CD4 and CD8 coreceptors that bind and effectively sequester the tyrosine kinase Lck, thereby preventing T cell receptor (TCR) signaling by non-MHC ligands that do not engage either coreceptor. This hypothesis predicts that, in coreceptor-deficient mice, alphabeta thymocytes would be signaled by non-MHC ligands to differentiate into alphabetaT cells lacking MHC specificity. We now report that MHC-independent alphabetaT cells were indeed generated in mice deficient in both coreceptors as well as MHC ("quad-deficient" mice) and that such mice contained a diverse alphabetaT cell repertoire whose MHC independence was confirmed at the clonal level. We conclude that CD4 and CD8 coreceptors impose MHC specificity on a broader alphabetaTCR repertoire during thymic selection by preventing thymocytes from being signaled by non-MHC ligands.

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model.

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.

Modulation of coreceptor transcription during positive selection dictates lineage fate independently of TCR/coreceptor specificity.

For developing T cells, coreceptor choice is matched to T cell antigen receptor (TCR) MHC specificity during positive selection in the thymus, but the mechanism remains uncertain. Here, we document that TCR-mediated positive selection signals inactivate the immature CD8(III) enhancer in double positive (DP) thymocytes, explaining in part the cessation of CD8 coreceptor transcription that occurs during positive selection. More importantly, by placing CD4 protein expression under the control of CD8 transcriptional regulatory elements, we demonstrate that cessation of CD4 coreceptor transcription during positive selection results in precisely the same lineage fate as cessation of CD8 coreceptor transcription. That is, MHC-II-signaled DP thymocytes differentiated into CD8-lineage cytotoxic T cells, despite the MHC-II specificity and CD4 dependence of their TCRs. This study demonstrates that termination of coreceptor transcription during positive selection promotes CD8-lineage fate, regardless of TCR specificity or coreceptor protein identity.

Exogenous IL-7 increases recent thymic emigrants in peripheral lymphoid tissue without enhanced thymic function.

Interleukin 7 (IL-7) is critical in maintaining thymic-dependent and thymic-independent pathways of T-cell homeostasis. T-cell receptor (TCR) rearrangement excision circles (TRECs) have been used as markers for recent thymic emigrants (RTEs) in assessing human thymic function. To study the thymic and peripheral effects of IL-7 on RTEs, we measured TREC content and peripheral naive T-cell subsets and turnover in IL-7-treated mice. Short-term administration of IL-7 into thymus-intact mice resulted in increased total TREC numbers, consistent with RTE accumulation. Decreases in TREC frequency were attributable to dilution secondary to increased cell turnover. Significantly, IL-7 administration into thymectomized mice resulted in patterns of decreased TREC frequency and increased total TREC number similar to those in IL-7-treated thymus-intact mice. Distinct patterns of naive cell and RTE distribution among peripheral immune organs and altered expression of CD11a were observed following IL-7 treatment in thymus-intact and thymectomized mice. These results demonstrate (1) that total TREC number and not TREC frequency accurately reflects quantitative changes in RTEs; (2) that short-term IL-7 administration results in preferential accumulations of RTEs among peripheral immune organs, accounting for the increase in TRECs in the total peripheral lymphoid pool; and (3) no evidence for regulation of thymic function by short-term IL-7 administration.

A dose effect of IL-7 on thymocyte development.

To study interleukin-7 (IL-7) in early thymocyte development, we generated mice transgenic (Tg) for the IL-7 gene under control of the lck proximal promoter. Founder line TgA, with the lowest level of IL-7 overexpression, showed enhanced alphabeta T-cell development. In contrast, in the highest overexpressing founder line, TgB, alphabeta T-cell development was disturbed with a block at the earliest intrathymic precursor stage. This was due to decreased progenitor proliferation as assessed by Ki-67 staining and in vivo bromodeoxyuridine (BrdU) incorporation. Bcl-2 was up-regulated in T-cell-committed progenitors in all Tg lines, and accounted for greater numbers of double positive (DP), CD4 single positive (SP), and CD8SP thymocytes in TgA mice where, in contrast to TgB mice, thymocyte progenitor proliferation was normal. Mixed marrow chimeras using TgB(+) and congenic mice as donors, and experiments using anti-IL-7 monoclonal antibody (MAb) in vivo, confirmed the role of IL-7 protein in the observed TgB phenotype. In conclusion, at low Tg overexpression, IL-7 enhanced alphabeta T-cell development by increasing thymocyte progenitor survival, while at high overexpression IL-7 reduces their proliferation, inducing a dramatic block in DP production. These results show for the first time in vivo a dose effect of IL-7 on alphabeta T-cell development and have implications for IL-7 in the clinical setting.

Unraveling a revealing paradox: Why major histocompatibility complex I-signaled thymocytes "paradoxically" appear as CD4+8lo transitional cells during positive selection of CD8+ T cells.

The mechanism by which T cell receptor specificity determines the outcome of the CD4/CD8 lineage decision in the thymus is not known. An important clue is the fact that major histocompatibility complex (MHC)-I-signaled thymocytes paradoxically appear as CD4+8lo transitional cells during their differentiation into CD8+ T cells. Lineage commitment is generally thought to occur at the CD4+8+ (double positive) stage of differentiation and to result in silencing of the opposite coreceptor gene. From this perspective, the appearance of MHC-I-signaled thymocytes as CD4+8lo cells would be due to effects on CD8 surface protein expression, not CD8 gene expression. But contrary to this perspective, this study demonstrates that MHC-I-signaled thymocytes appear as CD4+8lo cells because of transient down-regulation of CD8 gene expression, not because of changes in CD8 surface protein expression or distribution. This study also demonstrates that initial cessation of CD8 gene expression in MHC-I-signaled thymocytes is not necessarily indicative of commitment to the CD4+ T cell lineage, as such thymocytes retain the potential to differentiate into CD8+ T cells. These results challenge classical concepts of lineage commitment but fulfill predictions of the kinetic signaling model.

In vitro evidence that cytokine receptor signals are required for differentiation of double positive thymocytes into functionally mature CD8+ T cells.

CD4(+)8(+) double positive (DP) thymocytes differentiate into CD4(+) and CD8(+) mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8(+) T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4(-)8(+) thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8(+) thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8(+) T cells.

CD40 ligand functions non-cell autonomously to promote deletion of self-reactive thymocytes.

CD40 ligand (CD40L)-deficient mice have been shown to have a defect in negative selection of self-reactive T cells during thymic development. However, the mechanism by which CD40L promotes deletion of autoreactive thymocytes has not yet been elucidated. We have studied negative selection in response to endogenous superantigens in CD40L-deficient mice and, consistent with previous reports, have found a defect in negative selection in these mice. To test the requirement for expression of CD40L on T cells undergoing negative selection, we have generated chimeric mice in which CD40L wild-type and CD40L-deficient thymocytes coexist. We find that both CD40L wild-type and CD40L-deficient thymocytes undergo equivalent and efficient negative selection when these populations coexist in chimeric mice. These results indicate that CD40L can function in a non-cell-autonomous manner during negative selection. Deletion of superantigen-reactive thymocytes was normal in B7-1/B7-2 double-knockout mice, indicating that CD40-CD40L-dependent negative selection is not solely mediated by B7 up-regulation and facilitation of B7-dependent T cell signaling. Finally, although the absence of CD40-CD40L interactions impairs negative selection of autoreactive CD4(+) and CD8(+) cells during thymic development, we find that self-reactive T cells are deleted in the mature CD4(+) population through a CD40L-independent pathway.

Overview of flow cytometry.

This introductory unit provides an overview of terminology and techniques unique to flow cytometry and is divided into two sections. The first section discusses technical aspects of flow cytometry which apply primarily to the instrumentation-oriented flow cytometry phase. The second section discusses electronic cell separation using flow cytometry.