The role of Hedgehog signalling in the homeostasis and differentiation of GammaDelta and AlphaBeta T cells

The Hedgehog (Hh) signalling pathway is an important mediator of many mammalian developmental processes and mutations in Hh genes cause serious developmental disorders and birth defects in both mice and humans. The role of Hh signalling in the development of αβTCR+ cells has been, and continues to be, explored by our and other laboratories. However, the impact of Hh signalling on unconventional T cells is not well characterised. Here, we aim to investigate the effect of Hh signalling on thymic and peripheral γδ T cells, both in mice and humans. Our study reveals that key components of the Hh pathway are present in the murine thymus and spleen and that γδ T cells are responsive to Hh signalling. Furthermore, flow cytometry of mutant mice revealed that Sonic (Shh) and Desert (Dhh) hedgehog affect γδ T cell biology in distinct ways. Our research detected numerous changes in cell numbers, key cytokine production, subtype differentiation, peripheral localization in both fetal and adult tissues and in a LPS-induced disease model. In humans, we expanded γδ thymocytes and assessed their responsiveness to Hh signalling. Furthermore, we investigated the role of Indian hedgehog (Ihh) in the transition from CD4-CD8- double negative (DN) to CD4+CD8+ double positive (DP) stage during αβ T cell development. Analysis of mice with conditional deletion of Indian hedgehog showed that Ihh negatively regulates DN to DP transition of αβTCR+ cells. Crossing of this conditional knockout (KO) to the male-specific antigen HY indicated that this effect is related to TCR rearrangement and signalling and thymic selection. Analysis of fetal and adult thymi also demonstrated that Ihh plays a role in DP to single positive (SP) transition too. Analysis of hydrocortisone (HC)-treated heterozygous Ihh and RagKO conditional Ihh mice further elucidated the role of Ihh in αβ T cell development

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

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

Mutations in SNX14 cause a distinctive autosomal-recessive cerebellar ataxia and intellectual disability syndrome

Intellectual disability and cerebellar atrophy occur together in a large number of genetic conditions and are frequently associated with microcephaly and/or epilepsy. Here we report the identification of causal mutations in Sorting Nexin 14 (SNX14) found in seven affected individuals from three unrelated consanguineous families who presented with recessively inherited moderate-severe intellectual disability, cerebellar ataxia, early-onset cerebellar atrophy, sensorineural hearing loss, and the distinctive association of progressively coarsening facial features, relative macrocephaly, and the absence of seizures. We used homozygosity mapping and whole-exome sequencing to identify a homozygous nonsense mutation and an in-frame multiexon deletion in two families. A homozygous splice site mutation was identified by Sanger sequencing of SNX14 in a third family, selected purely by phenotypic similarity. This discovery confirms that these characteristic features represent a distinct and recognizable syndrome. SNX14 encodes a cellular protein containing Phox (PX) and regulator of G protein signaling (RGS) domains. Weighted gene coexpression network analysis predicts that SNX14 is highly coexpressed with genes involved in cellular protein metabolism and vesicle-mediated transport. All three mutations either directly affected the PX domain or diminished SNX14 levels, implicating a loss of normal cellular function. This manifested as increased cytoplasmic vacuolation as observed in cultured fibroblasts. Our findings indicate an essential role for SNX14 in neural development and function, particularly in development and maturation of the cerebellum