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.

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.

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.

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.

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.

Gli3 in fetal thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of Shh.

Gli3 is a Hedgehog (Hh)-responsive transcription factor that can function as a transcriptional repressor or activator. We show that Gli3 activity in mouse thymic epithelial cells (TECs) promotes positive selection and differentiation from CD4+ CD8+ to CD4+ CD8- single-positive (SP4) cells in the fetal thymus and that Gli3 represses Shh Constitutive deletion of Gli3, and conditional deletion of Gli3 from TECs, reduced differentiation to SP4, whereas conditional deletion of Gli3 from thymocytes did not. Conditional deletion of Shh from TECs increased differentiation to SP4, and expression of Shh was upregulated in the Gli3-deficient thymus. Use of a transgenic Hh reporter showed that the Hh pathway was active in thymocytes, and increased in the Gli3-deficient fetal thymus. Neutralisation of endogenous Hh proteins in the Gli3-/- thymus restored SP4 differentiation, indicating that Gli3 in TECs promotes SP4 differentiation by repression of Shh Transcriptome analysis showed that Hh-mediated transcription was increased whereas TCR-mediated transcription was decreased in Gli3-/- thymocytes compared with wild type.

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 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.

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.

Transplanted human thymus slices induce and support T-cell development in mice after cryopreservation.

Nude mouse human thymus transplant model: Fresh or cryopreserved and thawed human thymus slices were transplanted subcutaneously into recipient nude mice. Nude mice subsequently produced mouse CD3+ CD4+ T-cells.

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.

Thymus transplantation for complete DiGeorge syndrome: European experience.

BACKGROUND: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). METHODS: Twelve patients with cDGS underwent transplantation with allogeneic cultured thymus. OBJECTIVE: We sought to confirm and extend the results previously obtained in a single center. RESULTS: Two patients died of pre-existing viral infections without having thymopoiesis, and 1 late death occurred from autoimmune thrombocytopenia. One infant had septic shock shortly after transplantation, resulting in graft loss and the need for a second transplant. Evidence of thymopoiesis developed from 5 to 6 months after transplantation in 10 patients. Median circulating naive CD4 counts were 44 × 106/L (range, 11-440 × 106/L) and 200 × 106/L (range, 5-310 × 106/L) at 12 and 24 months after transplantation and T-cell receptor excision circles were 2,238/106 T cells (range, 320-8,807/106 T cells) and 4,184/106 T cells (range, 1,582-24,596/106 T cells). Counts did not usually reach normal levels for age, but patients were able to clear pre-existing infections and those acquired later. At a median of 49 months (range, 22-80 months), 8 have ceased prophylactic antimicrobials, and 5 have ceased immunoglobulin replacement. Histologic confirmation of thymopoiesis was seen in 7 of 11 patients undergoing biopsy of transplanted tissue, including 5 showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator expression was also demonstrated. Autoimmune complications were seen in 7 of 12 patients. In 2 patients early transient autoimmune hemolysis settled after treatment and did not recur. The other 5 experienced ongoing autoimmune problems, including thyroiditis (3), hemolysis (1), thrombocytopenia (4), and neutropenia (1). CONCLUSIONS: This study confirms the previous reports that thymus transplantation can reconstitute T cells in patients with cDGS but with frequent autoimmune complications in survivors.

The transcriptional activator Gli2 modulates T-cell receptor signalling through attenuation of AP-1 and NFκB activity.

Different tissues contain diverse and dynamic cellular niches, providing distinct signals to tissue-resident or migratory infiltrating immune cells. Hedgehog (Hh) proteins are secreted inter-cellular signalling molecules, which are essential during development and are important in cancer, post-natal tissue homeostasis and repair. Hh signalling mediated by the Hh-responsive transcription factor Gli2 also has multiple roles in T-lymphocyte development and differentiation. Here, we investigate the function of Gli2 in T-cell signalling and activation. Gene transcription driven by the Gli2 transcriptional activator isoform (Gli2A) attenuated T-cell activation and proliferation following T-cell receptor (TCR) stimulation. Expression of Gli2A in T-cells altered gene expression profiles, impaired the TCR-induced Ca(2+) flux and nuclear expression of NFAT2, suppressed upregulation of molecules essential for activation, and attenuated signalling pathways upstream of the AP-1 and NFκB complexes, leading to reduced activation of these important transcription factors. Inhibition of physiological Hh-dependent transcription increased NFκB activity upon TCR ligation. These data are important for understanding the molecular mechanisms of immunomodulation, particularly in tissues where Hh proteins or other Gli-activating ligands such as TGFß are upregulated, including during inflammation, tissue damage and repair, and in tumour microenvironments.

Sonic Hedgehog regulates thymic epithelial cell differentiation.

Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

Tissue-derived hedgehog proteins modulate Th differentiation and disease.

Genome-wide association studies of complex immune-mediated diseases have indicated that many genetic factors, each with individual low risk, contribute to overall disease. It is therefore timely and important to characterize how immune responses may be subtly modified by tissue context. In this article, we explore the role of tissue-derived molecules in influencing the function of T cells, which, owing to their migratory nature, come into contact with many different microenvironments through their lifespan. Hedgehog (Hh) proteins act as secreted morphogens, providing concentration-dependent positional and temporal cell-fate specification in solid tissues. Hh signaling is required for embryogenesis and is important in postnatal tissue renewal and in malignancy. However, the function of Hh in dynamic, fluid systems, such as in mammalian immunity, is largely unknown. In this article, we show that Hh-dependent transcription in T cells promoted Th2 transcriptional programs and differentiation, exacerbating allergic disease. Of interest, expression of Sonic Hh increased in lung epithelial cells following the induction of allergic disease, and lung T cells upregulated Hh target gene expression, indicating that T cells respond to locally secreted Hh ligands in vivo. We show that Il4, the key Th2 cytokine, is a novel transcriptional target of Hh signals in T cells, providing one mechanism for the role of Hh in Th differentiation. We propose that Hh, secreted from inflamed, remodeling, or malignant tissue, can modulate local T cell function. Our data present an unexpected and novel role for tissue-derived morphogens in the regulation of fluid immune responses, with implications for allergy and tumor responses, suggesting new uses for anti-Hh therapeutics.

Phosphorylation of NTRK1 at Y674/Y675 induced by TP53-dependent repression of PTPN6 expression: a potential novel prognostic marker for breast cancer.

We have identified a ligand-independent mechanism whereby the tumor suppressor, TP53, induces nerve growth factor receptor, NTRK1, phosphorylation at Y674/Y675 (NTRK1-pY674/pY675), via the repression of the NTRK1-phosphatase, PTPN6. This results in suppression of breast cancer cell proliferation. In this investigation, we aimed to establish whether perturbation of the wild-type TP53-NTRK1-pY674/pY675-PTPN6 pathway has an impact on disease-free survival of breast cancer patients without neo-adjuvant treatment. A total of 308 tumor samples were stained for NTRK1, NTRK1-pY674/pY675, PTPN6, and TP53 expression. Association between expression levels and disease-free survival was determined by the univariate/multivariate and Kaplan-Meir methods of analysis. DNA from tumors was sequenced to identify mutant or wild-type TP53. Tumors expressing NTRK1-pY674/pY675 but with undetectable or low levels of PTPN6 and TP53 were associated with prolonged 5, 10, and 15 years' disease-free survival by 48%, 36%, and 37%, respectively, in the multivariate analysis (P<0.05). A similar result was observed in tumors expressing wild-type TP53, NTRK1-pY674/pY675, and low or undetectable levels of PTPN6. Given that estrogen receptor-positive breast cancers encode wild-type TP53, we analyzed this expression pattern in these tumors. Multivariate analysis showed that it was significantly and independently predictive of prolonged survival by 66%, 70%, and 84%, respectively, (P<0.05). The Kaplan-Meir method demonstrated that NTRK1-pY674/pY675 together with undetectable or low levels of PTPN6 correlated with 59% probability of disease-free survival (median survival 15 years), compared with 7% probability of disease-free survival (median survival 4.5 years) when absent. In luminal A tumors, the presence of this pattern was estimated to have a 61% probability of disease-free survival (median survival 15 years), compared with 6% probability of disease-free survival (median survival 3 years) when it was absent. These results strongly suggest that expression of NTRK1-pY674/pY675 together with wild-type TP53 and low levels of PTPN6 expression are predictors of improved disease-free survival and that they could be useful biomarkers to predict clinical outcome.

Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog.

The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.

Role of Hedgehog signalling at the transition from double-positive to single-positive thymocyte.

In the thymus, developing T cells receive signals that determine lineage choice, specificity, MHC restriction and tolerance to self-antigen. One way in which thymocytes receive instruction is by secretion of Sonic hedgehog (Shh) from thymic epithelial cells. We have previously shown that Hedgehog (Hh) signalling in the thymus decreases the CD4:CD8 single-positive (SP) thymocyte ratio. Here, we present data indicating that double-positive (DP) thymocytes are Hh-responsive and that thymocyte-intrinsic Hh signalling plays a role in modulating the production of CD4(+) (SP4), CD8(+) (SP8) and unconventional T-cell subsets. Repression of physiological Hh signalling in thymocytes altered the proportions of DP and SP4 cells. Thymocyte-intrinsic Hh-dependent transcription also attenuated both the production of mature SP4 and SP8 cells, and the establishment of peripheral T-cell compartments in TCR-transgenic mice. Additionally, stimulation or withdrawal of Hh signals in the WT foetal thymus impaired or enhanced upregulation of the CD4 lineage-specific transcription factor Gata3 respectively. These data together suggest that Hh signalling may play a role in influencing the later stages of thymocyte development.

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.