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.

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.

Intramolecular polyspecificity in CD4 T-cell recognition of Ad-restricted epitopes of proteoglycan aggrecan.

T-cell recognition of MHC­peptide complexes shows a high degree of polyspecificity extending to recognition of a large number of structurally unrelated peptides. Examples of polyspecificity reported to date are confined to recognition of epitopes from distinct proteins or synthetic peptide libraries. Here we describe intramolecular polyspecificity of CD4 T cells specific for several epitopes within proteoglycan aggrecan, a structural glycoprotein of cartilage and candidate autoantigen in rheumatoid arthritis. T-cell hybridomas from aggrecan-immunized mice recognized four structurally unrelated epitopes from the G1 domain of aggrecan, but not other aggrecan epitopes or a variety of other peptide epitopes restricted by the same MHC class II allele. We also showed that the hierarchy of cross-reactivity broadly correlated with the strength of peptide binding to MHC class II. Similar polyspecificity was observed in responses of lymph node cells from peptide-immunized mice, suggesting polyspecificity of a significant proportion of the in vivo aggrecan specific T-cell repertoire. Polyspecific recognition of several epitopes within the same autoantigen may provide a novel mechanism to reach the activation threshold of low-affinity autoreactive T cells in the initiation of autoimmune diseases.

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.

Peptide-specific, TCR-alpha-driven, coreceptor-independent negative selection in TCR alpha-chain transgenic mice.

As thymocytes differentiate, Ag sensitivity declines, with immature CD4-CD8- double-negative (DN) cells being most susceptible to TCR signaling events. We show that expression of alphabetaTCR from the DN3 stage lowers the threshold for activation, allowing recognition of MHC peptides independently of the TCR beta-chain and without either T cell coreceptor. The MHC class I-restricted C6 TCR recognizes the Y-chromosome-derived Ag HYK(k)Smcy. Positive selection in C6 alphabetaTCR females is skewed to the CD8 compartment, whereas transgenic male mice exhibit early clonal deletion of thymocytes. We investigated the effect of the HYK(k)Smcy complex on developing thymocytes expressing the C6 TCR alpha-chain on a TCR-alpha(-/-) background. On the original selecting haplotype, the skew to the CD8 lineage is preserved. This is MHC dependent, as the normal bias to the CD4 subset is seen on an H2b background. In male H2k C6 alpha-only mice, the presence of the HYK(k)Smcy complex leads to a substantial deletion of thymocytes from the DN subset. This phenotype is replicated in H2k C6 alpha-only female mice expressing an Smcy transgene. Deletion is not dependent on the beta variable segment of the C6 TCR or on a restricted TCR-beta repertoire. In contrast, binding of HYK(k)Smcy and Ag-specific activation of mature CD8+ T cells is strictly dependent on the original C6 beta-chain. These data demonstrate that, in comparison with mature T cells, alphabetaTCR+ immature thymocytes can recognize and transduce signals in response to specific MHC-peptide complexes with relaxed binding requirements.

Non-obese diabetic mice select a low-diversity repertoire of natural regulatory T cells.

Thymus-derived Foxp3(+) natural regulatory CD4 T cells (nTregs) prevent autoimmunity through control of pathogenic, autoreactive T cells and other immune effector cells. Using T cell receptor (TCR) transgenic models, diversity within this lineage has been found to be similar to that of conventional CD4 T cells. To determine whether balanced TCR diversity may be perturbed in autoimmunity, we have analyzed receptor composition in C57BL/6 and autoimmune non-obese diabetic (NOD) mice. The natural regulatory and conventional CD4 repertoires of C57BL/6 had similar diversities. Despite the apparently normal thymic development of the NOD nTreg lineage, TCR diversity within the selected repertoire was markedly restricted. Detailed analysis of TCRalpha and -beta chain composition is consistent with positive selection into the natural regulatory lineage being under stringent audition for interaction with MHC class II/self-peptide. The NOD MHC region, including the unique H2-A(g7) class II molecule, partly accounts for the reduction in diversity, but additional NOD genetic contribution(s) are required for complete repertoire compaction. Mechanistic links between MHC, autoimmunity, and nTreg diversity identified in this study are discussed.

Sonic hedgehog negatively regulates pre-TCR-induced differentiation by a Gli2-dependent mechanism.

Hedgehog signaling regulates differentiation, survival, and proliferation of the earliest double-negative (DN) thymocytes, but its importance at later stages of T-cell development is controversial. Here we use loss- and gain-of-function mouse models to show that Shh, by signaling directly to the developing thymocyte, is a negative regulator of pre-TCR-induced differentiation from DN to double-positive (DP) cell. When hedgehog signaling was reduced, in the Shh(-/-) and Gli2(-/-) thymus, or by T lineage-specific transgenic expression of a transcriptional-repressor form of Gli2 (Gli2DeltaC(2)), differentiation to DP cell after pre-TCR signal transduction was increased. In contrast, when Hh signaling was constitutively activated in thymocytes, by transgenic expression of a constitutive transcriptional-activator form of Gli2 (Gli2DeltaN(2)), the production of DP cells was decreased. Gene expression profiling showed that physiologic Hh signaling in thymocytes maintains expression of the transcription factor FoxA2 on pre-TCR signal transduction.

The Gli3 transcription factor expressed in the thymus stroma controls thymocyte negative selection via Hedgehog-dependent and -independent mechanisms.

The Hedgehog (Hh) responsive transcription factor Gli3 is required for efficient thymocyte development in the fetus. In this study we show that Gli3, not detected in adult thymocytes, is expressed in the murine fetal and adult thymus stroma. PCR array analysis revealed Cxcl9, Rbp1, and Nos2 as novel target genes of Gli3. We show that Gli3 positively regulates the expression of these genes, most likely by suppressing an intermediate repressor. Deletion of autoreactive thymocytes depends on their interactions with the thymus stroma. Repression of the proapoptotic gene Nos2 in Gli3 mutants coincides with reduced apoptosis of double positive thymocytes undergoing negative selection in vitro and in vivo, and the production of autoreactive thymocytes. Taken together these data indicate that Gli3 controls thymocyte apoptosis and negative selection possibly via the regulation of Nos2. Defective Gli3 expression in the thymus stroma also resulted in decreased CD5 expression on mature thymocytes and inappropriate production of MHC class I-selected CD4(+) cells, both consistent with reduced TCR signal strength. Overall our data indicate that Gli3 expressed in the thymus stroma regulates negative selection and TCR signal strength via Hh-dependent and -independent mechanisms, with implications for autoimmunity.

Polymorphonuclear leucocyte phagocytic function, γδ T-lymphocytes and testosterone as separate stress-responsive markers of prolonged, high-intensity training programs.

Excessive exercise with limited recovery may lead to detrimental states of overreaching or the overtraining syndrome. Chronic maladaptation in endocrine and immune mechanisms occur with the incidence of these states. Exercise-induced cortisol and testosterone responses have been proposed as biomarkers of overreaching, with blunted responses following intensified-training periods. Yet, limited information on the effects of overreaching in immunity is available. Healthy individuals completed a 30-min running protocol (the RPETP) before and after a 12-day intensified-training period. Blood and saliva were collected before, after and 30min after RPETP at pre-training and post-training. Plasma and salivary cortisol and testosterone, leucocyte proliferation and polymorphonuclear leucocyte phagocytic activity were examined. Plasma and salivary cortisol were acutely unaffected pre-training (-14% and 0%, p â€‹> â€‹0.05) and post-training (-14% and +46%, p â€‹> â€‹0.05). Comparing pre-training with post-training, blunted responses were observed in plasma testosterone (43%-19%, p â€‹< â€‹0.05) and salivary testosterone (55%-24%, p â€‹> â€‹0.05). No acute or resting changes in total leucocyte counts or most leucocyte subsets occurred pre-training or post-training. Yet, a 194% acute elevation in γδ T-lymphocyte number occurred pre-training (p â€‹< â€‹0.05), and average resting concentrations were 174% higher post-training. Baseline phagocytic activity was 47% lower post-training (p â€‹< â€‹0.05). Intensified training was detrimental, significantly reducing phagocytic activity. Testosterone blunted post-training, indicating an excessive training-related hypothalamic-pituitary gonadal dysfunction. The γδ T-lymphocytes sensitivity to exercise was noted, rendering it as a potential stress-responsive cellular marker. The usefulness of the RPETP to track the onset of overreaching is proposed.

Frontline Science: Shh production and Gli signaling is activated in vivo in lung, enhancing the Th2 response during a murine model of allergic asthma.

The pathophysiology of allergic asthma is driven by Th2 immune responses after aeroallergen inhalation. The mechanisms that initiate, potentiate, and regulate airway allergy are incompletely characterized. We have shown that Hh signaling to T cells, via downstream Gli transcription factors, enhances T cell conversion to a Th2 phenotype. In this study, we showed for the first time, to our knowledge, that Gli-dependent transcription is activated in T cells in vivo during murine AAD, a model for the immunopathology of asthma, and that genetic repression of Gli signaling in T cells decreases the differentiation and recruitment of Th2 cells to the lung. T cells were not the only cells that expressed activated Gli during AAD. A substantial proportion of eosinophils and lung epithelial cells, both central mediators of the immunopathology of asthma, also underwent Hh/Gli signaling. Finally, Shh increased Il-4 expression in eosinophils. We therefore propose that Hh signaling during AAD is complex, involving multiple cell types, signaling in an auto- or paracrine fashion. Improved understanding of the role of this major morphogenetic pathway in asthma may give rise to new drug targets for this chronic condition.

Direct BMP2/4 signaling through BMP receptor IA regulates fetal thymocyte progenitor homeostasis and differentiation to CD4+CD8+ double-positive cell.

BMP2/4 signaling is required for embryogenesis and involved in thymus morphogenesis and T-lineage differentiation. In vitro experiments have shown that treatment of thymus explants with exogenous BMP4 negatively regulated differentiation of early thymocyte progenitors and the transition from CD4-CD8- (DN) to CD4+CD8+ (DP). Here we show that in vivo BMP2/4 signaling is required for fetal thymocyte progenitor homeostasis and expansion, but negatively regulates differentiation from DN to DP cell. Unexpectedly, conditional deletion of BMPRIA from fetal thymocytes (using the Cre-loxP system and directing excision to hematopoietic lineage cells with the Vav promoter) demonstrated that physiological levels of BMP2/4 signaling directly to thymocytes through BMPRIA are required for normal differentiation and expansion of early fetal DN thymocytes. In contrast, the arrest in early thymocyte progenitor differentiation caused by exogenous BMP4 treatment of thymus explants is induced in part by direct signaling to thymocytes through BMPRIA, and in part by indirect signaling through non-hematopoietic cells. Analysis of the transition from fetal DN to DP cell, both by ex vivo analysis of conditional BMPRIA-deficient thymocytes and by treatment of thymus explants with the BMP4-inhibitor Noggin demonstrated that BMP2/4 signaling is a negative regulator at this stage. We showed that at this stage of fetal T-cell development BMP2/4 signals directly to thymocytes through BMPRIA.

T-cell reconstitution after thymus xenotransplantation induces hair depigmentation and loss.

Here we present a mouse model for T-cell targeting of hair follicles, linking the pathogenesis of alopecia to that of depigmentation disorders. Clinically, thymus transplantation has been successfully used to treat T-cell immunodeficiency in congenital athymia, but is associated with autoimmunity. We established a mouse model of thymus transplantation by subcutaneously implanting human thymus tissue into athymic C57BL/6 nude mice. These xenografts supported mouse T-cell development. Surprisingly, we did not detect multiorgan autoimmune disease. However, in all transplanted mice, we noted a striking depigmentation and loss of hair follicles. Transfer of T cells from transplanted nudes to syngeneic black-coated RAG(-/-) recipients caused progressive, persistent coat-hair whitening, which preceded patchy hair loss in depigmented areas. Further transfer experiments revealed that these phenomena could be induced by CD4+ T cells alone. Immunofluorescent analysis suggested that Trp2+ melanocyte-lineage cells were decreased in depigmented hair follicles, and pathogenic T cells upregulated activation markers when exposed to C57BL/6 melanocytes in vitro, suggesting that these T cells are not tolerant to self-melanocyte antigens. Our data raise interesting questions about the mechanisms underlying tissue-specific tolerance to skin antigens.

Role of endogenous annexin-A1 in the regulation of thymocyte positive and negative selection.

We have recently shown that endogenous Annexin-A1 (AnxA1) plays a homeostatic regulatory role in mature T cells by modulating the strength of TCR signaling. In this study we investigated the role of endogenous AnxA1 in thymocyte maturation. Analysis of AnxA1(-/-) thymocyte populations at the immature CD4(-)CD8(-) double negative (DN) stage showed a proportional decrease in the DN1 and an increase in the DN3 subsets compared to control littermates. There were no significant differences in thymocyte numbers or proportions of CD4(+) and CD8(+) single positive (SP) populations between Anx1(-/-) and AnxA1(+/+) mice. However, when we crossed AnxA1(-/-) mice onto HY-TCR transgenic mice, we observed an increase in CD4(+)CD8(+) double positive (DP) and CD4 SP cells in male AnxA1(-/-)/HY-TCR compared to AnxA1(+/+)/HY-TCR. Conversely, female AnxA1(-/-)/HY-TCR mice showed an increase in DP and a decrease in CD8 (SP) cells compared to female AnxA1(+/+)/HY-TCR. Biochemical analysis of the signaling pathways responsible for these effects showed a decrease in anti-CD3-induced Erk phosphorylation and NFkappaB activation in AnxA1(-/-) thymocytes compared to control littermates. Together these findings demonstrate a role for endogenous AnxA1 in regulating both positive and negative selection of the TCR repertoire. These results suggest that targeting AnxA1 expression or function in T cells could represent a useful approach for the development of novel therapies for the treatment of autoimmune diseases.

Non-redundant role for the transcription factor Gli1 at multiple stages of thymocyte development.

The Hedgehog (Hh) signaling pathway influences multiple stages of murine T-cell development. Hh signaling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse development and can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and nonredundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4⁻CD8⁻ double negative (DN) thymocytes before pre- TCR signal transduction, and a negative regulatory function after pre-TCR signaling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage.

Public T cell receptor beta-chains are not advantaged during positive selection.

Studies of human and murine T cells have shown that public TCR beta-chain rearrangements can dominate the Ag-specific and naive repertoires of distinct individuals. We show that mouse T cells responding to the minor histocompatibility Ag HYDbSmcy share an invariant Vbeta8.2-Jbeta2.3 TCR gene rearrangement. The dominance of this rearrangement shows that it successfully negotiated thymic selection and was highly favored during clonal expansion in all animals examined. We hypothesized that such beta-chains are advantaged during thymic and/or peripheral selection and, as a result, may be over-represented in the naive repertoire. A sequencing study was undertaken to examine the diversity of Vbeta8.2-Jbeta2.3 CDR3 loops from naive T cell repertoires of multiple mice. Public TCR beta-chain sequences were identified across different repertoires and MHC haplotypes. To determine whether such public beta-chains are advantaged during thymic selection, individual chains were followed through T cell development in a series of novel bone marrow competition chimeras. We demonstrate that beta-chains were positively selected with similar efficiency regardless of CDR3 loop sequence. Therefore, the establishment and maintenance of public beta-chains in the periphery is predominantly controlled by post-thymic events through modification of the primary, thymus-derived TCR repertoire.