Cryopreservation of mouse thymus depletes thymocytes but supports immune reconstitution on transplantation.

Cryopreservation of mouse thymus depletes donor thymocytes but preserves thymus function when transplanted after thawing into athymic mice. No differences in immune reconstitution were observed between fresh and frozen/thawed transplants suggesting that donor thymocyte depletion does not affect outcome. Thus, cryopreservation of thymus may improve outcomes in thymus transplant patients.

Hedgehog signalling in allograft vasculopathy: a new therapeutic target?

Allograft vasculopathy (AV) leads to chronic rejection of organ transplants, but its causes are obscure. New research from the Jane-Wit laboratory showed that Sonic Hedgehog (SHH) signalling from damaged graft endothelium drives vasculopathy by promoting proinflammatory cytokine production and NLRP3-inflammasome activation in alloreactive CD4+PTCH1hiPD-1hiT memory cells, offering new diagnostic and therapeutic strategies.

The Pioneer Transcription Factor Foxa2 Modulates T Helper Differentiation to Reduce Mouse Allergic Airway Disease.

Foxa2, a member of the Forkhead box (Fox) family of transcription factors, plays an important role in the regulation of lung function and lung tissue homeostasis. FOXA2 expression is reduced in the lung and airways epithelium of asthmatic patients and in mice absence of Foxa2 from the lung epithelium contributes to airway inflammation and goblet cell hyperplasia. Here we demonstrate a novel role for Foxa2 in the regulation of T helper differentiation and investigate its impact on lung inflammation. Conditional deletion of Foxa2 from T-cells led to increased Th2 cytokine secretion and differentiation, but decreased Th1 differentiation and IFN-γ expression in vitro. Induction of mouse allergic airway inflammation resulted in more severe disease in the conditional Foxa2 knockout than in control mice, with increased cellular infiltration to the lung, characterized by the recruitment of eosinophils and basophils, increased mucus production and increased production of Th2 cytokines and serum IgE. Thus, these experiments suggest that Foxa2 expression in T-cells is required to protect against the Th2 inflammatory response in allergic airway inflammation and that Foxa2 is important in T-cells to maintain the balance of effector cell differentiation and function in the lung.

Anti-CCR9 chimeric antigen receptor T cells for T-cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes, associated with higher rates of induction failure compared with those in B cell acute lymphoblastic leukemia. The potent immunotherapeutic approaches applied in B cell acute lymphoblastic leukemia, which have revolutionized the treatment paradigm, have proven more challenging in T-ALL, largely due to a lack of target antigens expressed on malignant but not healthy T cells. Unlike B cell depletion, T-cell aplasia is highly toxic. Here, we show that the chemokine receptor CCR9 is expressed in >70% of cases of T-ALL, including >85% of relapsed/refractory disease, and only on a small fraction (<5%) of normal T cells. Using cell line models and patient-derived xenografts, we found that chimeric antigen receptor (CAR) T-cells targeting CCR9 are resistant to fratricide and have potent antileukemic activity both in vitro and in vivo, even at low target antigen density. We propose that anti-CCR9 CAR-T cells could be a highly effective treatment strategy for T-ALL, avoiding T cell aplasia and the need for genome engineering that complicate other approaches.

Sonic Hedgehog signalling in the regulation of barrier tissue homeostasis and inflammation.

Epithelial barrier tissues such as the skin and airway form an essential interface between the mammalian host and its external environment. These physical barriers are crucial to prevent damage and disease from environmental insults and allergens. Failure to maintain barrier function against such risks can lead to severe inflammatory disorders, including atopic dermatitis and asthma. Here, we discuss the role of the morphogen Sonic Hedgehog in postnatal skin and lung and the impact of Shh signalling on repair, inflammation, and atopic disease in these tissues.

Metabolite and thymocyte development defects in ADA-SCID mice receiving enzyme replacement therapy.

Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme intrinsic to the purine salvage pathway, leads to severe combined immunodeficiency (SCID) both in humans and mice. Lack of ADA results in the intracellular accumulation of toxic metabolites which have effects on T cell development and function. While untreated ADA-SCID is a fatal disorder, there are different therapeutic options available to restore ADA activity and reconstitute a functioning immune system, including enzyme replacement therapy (ERT). Administration of ERT in the form of pegylated bovine ADA (PEG-ADA) has proved a life-saving though non-curative treatment for ADA-SCID patients. However, in many patients treated with PEG-ADA, there is suboptimal immune recovery with low T and B cell numbers. Here, we show reduced thymus cellularity in ADA-SCID mice despite weekly PEG-ADA treatment. This was associated with lack of effective adenosine (Ado) detoxification in the thymus. We also show that thymocyte development in ADA-deficient thymi is arrested at the DN3-to-DN4 stage transition with thymocytes undergoing dATP-induced apoptosis rather than defective TCRß rearrangement or ß-selection. Our studies demonstrate at a detailed level that exogenous once-a-week enzyme replacement does not fully correct intra-thymic metabolic or immunological abnormalities associated with ADA deficiency.

Systemic Pharmacological Smoothened Inhibition Reduces Lung T-Cell Infiltration and Ameliorates Th2 Inflammation in a Mouse Model of Allergic Airway Disease.

Allergic asthma is a common inflammatory airway disease in which Th2 immune response and inflammation are thought to be triggered by inhalation of environmental allergens. Many studies using mouse models and human tissues and genome-wide association have indicated that Sonic Hedgehog (Shh) and the Hedgehog (Hh) signaling pathway are involved in allergic asthma and that Shh is upregulated in the lung on disease induction. We used a papain-induced mouse model of allergic airway inflammation to investigate the impact of systemic pharmacological inhibition of the Hh signal transduction molecule smoothened on allergic airway disease induction and severity. Smoothened-inhibitor treatment reduced the induction of Shh, IL-4, and IL-13 in the lung and decreased serum IgE, as well as the expression of Smo, Il4, Il13, and the mucin gene Muc5ac in lung tissue. Smoothened inhibitor treatment reduced cellular infiltration of eosinophils, mast cells, basophils, and CD4+ T-cells to the lung, and eosinophils and CD4+ T-cells in the bronchoalveolar lavage. In the mediastinal lymph nodes, smoothened inhibitor treatment reduced the number of CD4+ T-cells, and the cell surface expression of Th2 markers ST2 and IL-4rα and expression of Th2 cytokines. Thus, overall pharmacological smoothened inhibition attenuated T-cell infiltration to the lung and Th2 function and reduced disease severity and inflammation in the airway.

The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during thymocyte positive selection.

During positive selection at the transition from CD4+CD8+ double-positive (DP) to single-positive (SP) thymocyte, TCR signalling results in appropriate MHC restriction and signals for survival and progression. We show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection of mouse T cells and that Foxa1 and Foxa2 have overlapping/compensatory roles. Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes reduced positive selection and development of CD4SP, CD8SP and peripheral naïve CD4+ T cells. Foxa1 and Foxa2 regulated the expression of many genes encoding splicing factors and regulators, including Mbnl1, H1f0, Sf3b1, Hnrnpa1, Rnpc3, Prpf4b, Prpf40b and Snrpd3. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated in the double Foxa1/Foxa2 conditional knockout, leading to >850 differentially used exons. Many genes important for this stage of T-cell development (Ikzf1-3, Ptprc, Stat5a, Stat5b, Cd28, Tcf7) and splicing factors (Hnrnpab, Hnrnpa2b1, Hnrnpu, Hnrnpul1, Prpf8) showed multiple differentially used exons. Thus, Foxa1 and Foxa2 are required during positive selection to regulate alternative splicing of genes essential for T-cell development, and, by also regulating splicing of splicing factors, they exert widespread control of alternative splicing.

T cell phenotype in paediatric heart transplant recipients.

Paediatric heart transplantation recipients suffer an increased incidence of infectious, autoimmune and allergic problems. The relative roles of thymus excision and immunosuppressive treatments in contributing to these sequelae are not clear. We compared the immunological phenotypes of 25 heart transplant recipients (Tx), 10 children who underwent thymus excision during non-transplantation cardiac surgery (TE) and 25 age range-matched controls, in two age bands: 1-9 and 10-16 years. Significant differences from controls were seen mainly in the younger age band with Tx showing lower CD3 and CD4 cell counts whilst TE showed lower CD8 cell counts. Naïve T cell and recent thymic emigrant proportions and counts were significantly lower than controls in both groups in the lower age band. T cell recombination excision circle (TREC) levels were lower than controls in both groups in both age bands. There were no differences in regulatory T cells, but in those undergoing thymus excision in infancy, their proportions were higher in TE than Tx, a possible direct effect of immunosuppression. T cell receptor V beta spectratyping showed fewer peaks in both groups than in controls (predominantly in the older age band). Thymus excision in infancy was associated with lower CD8 cell counts and higher proportions of Tregs in TE compared to Tx. These data are consistent with thymus excision, particularly in infancy, being the most important influence on immunological phenotype after heart transplantation.

The transcriptional repressor Bcl6 promotes pre-TCR-induced thymocyte differentiation and attenuates Notch1 activation.

Pre-T-cell receptor (TCR) signal transduction is required for developing thymocytes to differentiate from CD4-CD8- double-negative (DN) cell to CD4+CD8+ double-positive (DP) cell. Notch signalling is required for T-cell fate specification and must be maintained throughout ß-selection, but inappropriate Notch activation in DN4 and DP cells is oncogenic. Here, we show that pre-TCR signalling leads to increased expression of the transcriptional repressor Bcl6 and that Bcl6 is required for differentiation to DP. Conditional deletion of Bcl6 from thymocytes reduced pre-TCR-induced differentiation to DP cells, disrupted expansion and enrichment of intracellular TCRß+ cells within the DN population and increased DN4 cell death. Deletion also increased Notch1 activation and Notch-mediated transcription in the DP population. Thus, Bcl6 is required in thymocyte development for efficient differentiation from DN3 to DP and to attenuate Notch1 activation in DP cells. Given the importance of inappropriate NOTCH1 signalling in T-cell acute lymphoblastic leukaemia (T-ALL), and the involvement of BCL6 in other types of leukaemia, this study is important to our understanding of T-ALL.

The IFITM protein family in adaptive immunity.

Interferon-inducible transmembrane (IFITM) proteins are a family of small homologous proteins, localized in the plasma and endolysosomal membranes, which confer cellular resistance to many viruses. In addition, several distinct functions have been associated with different IFITM family members, including germ cell specification (IFITM1-IFITM3), osteoblast function and bone mineralization (IFITM5) and immune functions (IFITM1-3, IFITM6). IFITM1-3 are expressed by T cells and recent experiments have shown that the IFITM proteins are directly involved in adaptive immunity and that they regulate CD4+ T helper cell differentiation in a T-cell-intrinsic manner. Here we review the role of the IFITM proteins in T-cell differentiation and function.

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.

Sonic Hedgehog signaling limits atopic dermatitis via Gli2-driven immune regulation.

Hedgehog (Hh) proteins regulate development and tissue homeostasis, but their role in atopic dermatitis (AD) remains unknown. We found that on induction of mouse AD, Sonic Hedgehog (Shh) expression in skin, and Hh pathway action in skin T cells were increased. Shh signaling reduced AD pathology and the levels of Shh expression determined disease severity. Hh-mediated transcription in skin T cells in AD-induced mice increased Treg populations and their suppressive function through increased active transforming growth factor-ß (TGF-ß) in Tregs signaling to skin T effector populations to reduce disease progression and pathology. RNA sequencing of skin CD4+ T cells from AD-induced mice demonstrated that Hh signaling increased expression of immunoregulatory genes and reduced expression of inflammatory and chemokine genes. Addition of recombinant Shh to cultures of naive human CD4+ T cells in iTreg culture conditions increased FOXP3 expression. Our findings establish an important role for Shh upregulation in preventing AD, by increased Gli-driven Treg cell-mediated immune suppression, paving the way for a potential new therapeutic strategy.

Dysregulated gene expression in oocysts of Plasmodium berghei LAP mutants.

Malaria parasite oocysts generate sporozoites by a process termed sporogony. Essential for successful sporogony of Plasmodium berghei in Anopheles stephensi mosquitoes is a complex of six LCCL lectin domain adhesive-like proteins (LAPs). LAP null mutant oocysts undergo growth and mitosis but fail to form sporozoites. At a cytological level, LAP null mutant oocyst development is indistinguishable from its wildtype counterparts for the first week, supporting the hypothesis that LAP null mutant oocysts develop normally before cytokinesis. We show here that LAP1 null mutant oocysts display highly reduced expression of sporozoite proteins and their transcription factors. Our findings indicate that events leading up to the cytokinesis defect in LAP null mutants occur early in oocyst development.

IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation.

The interferon-inducible transmembrane (Ifitm/Fragilis) genes encode homologous proteins that are induced by IFNs. Here, we show that IFITM proteins regulate murine CD4+ Th cell differentiation. Ifitm2 and Ifitm3 are expressed in wild-type (WT) CD4+ T cells. On activation, Ifitm3 was downregulated and Ifitm2 was upregulated. Resting Ifitm-family-deficient CD4+ T cells had higher expression of Th1-associated genes than WT and purified naive Ifitm-family-deficient CD4+ T cells differentiated more efficiently to Th1, whereas Th2 differentiation was inhibited. Ifitm-family-deficient mice, but not Ifitm3-deficient mice, were less susceptible than WT to induction of allergic airways disease, with a weaker Th2 response and less severe disease and lower Il4 but higher Ifng expression and IL-27 secretion. Thus, the Ifitm family is important in adaptive immunity, influencing Th1/Th2 polarization, and Th2 immunopathology.

Foxa1 and Foxa2 in thymic epithelial cells (TEC) regulate medullary TEC and regulatory T-cell maturation.

The Foxa1 and Foxa2 transcription factors are essential for mouse development. Here we show that they are expressed in thymic epithelial cells (TEC) where they regulate TEC development and function, with important consequences for T-cell development. TEC are essential for T-cell differentiation, lineage decisions and repertoire selection. Conditional deletion of Foxa1 and Foxa2 from murine TEC led to a smaller thymus with a greater proportion of TEC and a greater ratio of medullary to cortical TEC. Cell-surface MHCI expression was increased on cortical TEC in the conditional Foxa1Foxa2 knockout thymus, and MHCII expression was reduced on both cortical and medullary TEC populations. These changes in TEC differentiation and MHC expression led to a significant reduction in thymocyte numbers, reduced positive selection of CD4+CD8+ cells to the CD4 lineage, and increased CD8 cell differentiation. Conditional deletion of Foxa1 and Foxa2 from TEC also caused an increase in the medullary TEC population, and increased expression of Aire, but lower cell surface MHCII expression on Aire-expressing mTEC, and increased production of regulatory T-cells. Thus, Foxa1 and Foxa2 in TEC promote positive selection of CD4SP T-cells and modulate regulatory T-cell production and activity, of importance to autoimmunity.

A temporally dynamic Foxp3 autoregulatory transcriptional circuit controls the effector Treg programme.

Regulatory T cells (Treg) are negative regulators of the immune response; however, it is poorly understood whether and how Foxp3 transcription is induced and regulated in the periphery during T-cell responses. Using Foxp3-Timer of cell kinetics and activity (Tocky) mice, which report real-time Foxp3 expression, we show that the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased during inflammation. These persistent dynamics of Foxp3 transcription determine the effector Treg programme and are dependent on a Foxp3 autoregulatory transcriptional circuit. Persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules, including CTLA-4 and effector Treg signature genes. Using RNA-seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof of principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti-TNFRII antibody, and high-frequency Foxp3 expressors are targeted by anti-OX40 antibody. Collectively, our study dissects time-dependent mechanisms behind Foxp3-driven T-cell regulation and establishes the Foxp3-Tocky system as a tool to investigate the mechanisms behind T-cell immunotherapies.

A timer for analyzing temporally dynamic changes in transcription during differentiation in vivo.

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyze in vivo. We establish a new tool, Timer of cell kinetics and activity (Tocky; or toki [time in Japanese]). Tocky uses the fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue to red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR) signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.