The use of genomewide ENU mutagenesis screens to unravel complex mammalian traits: Identifying genes that regulate organ-specific and systemic autoimmunity

T-cell development is perhaps one of the best understood processes of mammalian cell differentiation, as many of the genes and pathways have been identified. By contrast, relatively little is known about the genes and pathways involved in immunological tolerance to self-antigens. Here, we describe the challenges associated with a genomewide screen designed at identifying new immune regulatory genes that uses a model of organ-specific autoimmunity leading to type 1 diabetes. The successful propagation and identification of the new gene variants will shed light on the various developmental checkpoints in lymphocyte development that are crucial for establishing tolerance to self-antigens

Consequences of increased CD45RA and RC isoforms for TCR signaling and peripheral T cell deficiency resulting from heterogeneous nuclear ribonucleoprotein L-like mutation

CD45 is the most abundant protein tyrosine phosphatase in the plasma membrane of T cells and serves a critical role in TCR signaling. Different CD45 isoforms are made by alternative mRNA splicing depending on the stage of T cell development and activation, yet their role remains unclear. Expression of CD45RA and RC isoforms is increased 20- to 200-fold on T cells from thunder mice with a loss-of-function mutation in the RNA-binding protein, heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL), although total CD45 expression is unaltered. In this study, we test the hypothesis that this shift in CD45 isoform expression alters TCR signaling, thymic selection, and accumulation of peripheral T cells. There was no discernable effect of the change in CD45 isoform expression upon Lck phosphorylation or T cell positive and negative selection, whereas these indices were strongly affected by a decrease in the overall amount of CD45 in Ptprc mutant animals. The one exception to this conclusion was in thymocytes from Ptprcloc/loc animals with 4% of normal CD45 protein levels, where Lck505 phosphorylation was increased 25% in Hnrpll mutant cells, suggesting that high m.w. CD45 isoforms had lower Lck505 phosphatase activity in this context. In T cells with no CD45 protein, hnRNPLL mutation still diminished peripheral T cell accumulation, demonstrating that hnRNPLL regulates T cell longevity independently from its effects on CD45 splicing

Differential Requirement for the CD45 Splicing Regulator hnRNPLL for Accumulation of NKT and Conventional T Cells

Natural killer T (NKT) cells represent an important regulatory T cell subset that develops in the thymus and contains immature (NK1.1lo) and mature (NK1.1hi) cell subsets. Here we show in mice that an inherited mutation in heterogeneous ribonucleoprotein L-like protein (hnRNPLLthunder), that shortens the survival of conventional T cells, has no discernible effect on NKT cell development, homeostasis or effector function. Thus, Hnrpll deficiency effectively increases the NKT∶T cell ratio in the periphery. However, Hnrpll mutation disrupts CD45RA, RB and RC exon silencing of the Ptprc mRNA in both NKT and conventional T cells, and leads to a comparably dramatic shift to high molecular weight CD45 isoforms. In addition, Hnrpll mutation has a cell intrinsic effect on the expression of the developmentally regulated cell surface marker NK1.1 on NKT cells in the thymus and periphery but does not affect cell numbers. Therefore our results highlight both overlapping and divergent roles for hnRNPLL between conventional T cells and NKT cells. In both cell subsets it is required as a trans-acting factor to regulate alternative splicing of the Ptprc mRNA, but it is only required for survival of conventional T cells

Cooperation between somatic Ikaros and Notch1 mutations at the inception of T-ALL

To understand the interactions between Notch1 and Ikaros in the evolution of T cell acute lymphoblastic leukemia (T-ALL), we traced the evolution of T-ALL in mice with an inherited Ikaros mutation, Ikzf1Plstc which inactivates DNA binding. DNA-binding Ikaros repressed Notch1 response in transfected cell lines and in CD4+8+ (DP) thymocytes from young pre-leukemic Ikzf1Plstc heterozygous mice. In DP thymocytes, a 50-1000 fold escalation in mRNA for Notch1 target genes Hes1 and Dtx1 preceded thymic lymphoma or leukemia and was closely correlated with the first detectable differentiation abnormalities loss of heterozygosity (LOH) eliminating wild-type Ikzf1, and multiple missense and truncating Notch1 mutations. These findings illuminate the early stages of leukemogenesis by demonstrating progressive exaggeration of Notch1 responsiveness at the DP thymocyte stage brought about by multiple mutations acting in concert upon the Notch1 pathwa

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to Type 1 Diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against Type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially auto-aggressive CD4+ T cells. Here we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme antigen expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4+ T cells in pre-diabetic 3A9-TCR x insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency because it does not affect thymic negative selection of islet-reactive CD4+ cells nor the numbers of islet-specific CD4+ or CD4+ FOXP3+ T cells in the periphery, although it decreased differentiation of inducible Treg (iTreg) cells from TGF-b treated 3A9-TCR cells in vitro. When removed from Tregs and placed in culture, Cblb-deficient islet-reactive CD4+ cells reveal a capacity to proliferate to HEL antigen that is repressed in wild-type cells. This latent failure of T cell anergy is nevertheless controlled in vivo in pre-diabetic mice, so that islet-reactive CD4+ cells in spleen and pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb-deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wks of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance, and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity

Loss of Roquin induces early death and immune deregulation but not autoimmunity

Deletion of Roquin in T or B cells, or in the entire hematopoietic system of mice, alters immune homeostasis but does not result in autoimmunity

Development and validation of the Australian Aboriginal racial identity and self-esteem survey for 8-12 year old children (IRISE-C)

Introduction: In Australia, there is little empirical research of the racial identity of Indigenous children and youth as the majority of the current literature focuses on adults. Furthermore, there are no instruments developed with cultural appropriateness when exploring the identity and self-esteem of the Australian Aboriginal population, especially children. The IRISE-C (Racial Identity and Self-Esteem of children) inventory was developed to explore the elements of racial identity and self-esteem of urban, rural and regional Aboriginal children. This paper describes the development and validation of the IRISE-C instrument with over 250 Aboriginal children aged 8 to 12 years. Methods: A pilot of the IRISE C instrument was combined with individual interviews and was undertaken with 35 urban Aboriginal children aged 8-12 years. An exploratory factor analysis was performed to refine the survey and reduce redundant items in readiness for the main study. In the main study, the IRISE C was employed to 229 Aboriginal children aged 6-13 years across three sites (rural, regional and urban) in Western Australia. An exploratory factor analysis using Principal axis factoring was used to assess the fit of items and survey structure. A confirmatory factor analysis was then employed using LISREL (diagonally weighted least squares) to assess factor structures across domains. Internal consistency and reliability of subscales were assessed using Cronbach's co-efficient alpha. Results: The pilot testing identified two key concepts - children's knowledge of issues related to their racial identity, and the importance, or salience, that they attach to these issues. In the main study, factor analyses showed two clear factors relating to: Aboriginal culture and traditions; and a sense of belonging to an Aboriginal community. Principal Axis Factoring of the Knowledge items supported a 2-factor solution, which explained 38.7 % of variance. Factor One (Aboriginal culture) had a Cronbach's alpha of 0.835; Factor 2 (racial identity) had a Cronbach's alpha of 0.800, thus demonstrating high internal reliability of the scales. Conclusion: The IRISE-C has been shown to be a valid instrument useful of exploring the development of racial identity of Australian Aboriginal children across the 8-12 year old age range and across urban, rural and regional geographical locations

Conflito trabalho-família, auto eficácia parental e estilos parentais percebidos em pais e mães da cidade de Talca no Chile

The relationship between levels of work-family conflict, parental self-efficacy and perceived parenting styles in a group of 43 school children and both working parents is analyzed, controlling for socio-demographic variables. Also, gender differences are identified in the variables and the relationship between them in relation to the number of children. Three instruments were applied to the sample for measuring the referred variables. A significant and negative relationship is observed between levels of work-family conflict and parental self-efficacy (r = -0.484, P <0.001). The authoritarian parenting style has greater association with self-efficacy (r = 0.301, P = 0.005). A significant and negative relationship between self-efficacy and number of children (r = -0.257, P = 0.017) is reported. Finally, it is concluded that women have greater work-family conflict than men.Se analiza la relación existente entre los niveles de conflicto trabajo-familia, autoeficacia parental y estilos parentales percibidos en un grupo de 43 niños estudiantes y ambos padres trabajadores, controlando las variables sociodemográficas. Así mismo, se identifican las diferencias por género en las variables, y la relación que existe entre ellas con respecto al número de hijos. A la muestra le fueron aplicados tres instrumentos de medición de las variables referidas. Se observa una relación significativa y negativa entre los niveles de conflicto trabajo-familia y la autoeficacia parental (r= -0,484; p<0,001). El estilo parental autoritario presenta mayor asociación con autoeficacia (r=0,301; p=0,005). Se reporta una relación significativa y negativa entre autoeficacia y número de hijos (r=-0,257; p=0,017). Finalmente se reporta que las mujeres presentan mayor conflicto trabajo-familia que los hombres.Analisa-se a relação existente entre os níveis de conflito trabalho-família, auto eficácia parental e estilos parentais percebidos em um grupo de 43 crianças estudantes de pais trabalhadores, controlando as variáveis sociodemográficas. Assim mesmo, identificam-se as diferenças por gênero nas variáveis, e a relação que existe entre elas com respeito ao número de filhos. Foram aplicados à mostra três instrumentos de medição das variáveis referidas. Observa-se uma relação significativa e negativa entre os níveis de conflito trabalho-família e a auto eficácia parental (r= -0,484; p<0,001). O estilo parental autoritário apresenta maior associação com autoeficácia (r=0,301; p=0,005). Reporta-se uma relação significativa e negativa entre autoeficácia e número de filhos (r=-0,257; p=0,017). Finalmente reporta-se que as mulheres apresentam maior conflito trabalho-família que os homens

Predicting Filipino Mothers\u27 and Fathers\u27 Reported Use of Corporal Punishment From Education, Authoritarian Attitudes, and Endorsement of Corporal Punishment

The relations of education, authoritarian childrearing attitudes, and endorsement of corporal punishment to Filipino parents\u27 reported use of corporal punishment were examined using two waves of data. Structured interviews using self-report questionnaires were conducted with 117 mothers and 98 fathers from 120 families when their children were 8 years old, and when their children were 9 years old. Path analyses showed that, among mothers, higher education predicted lower authoritarian attitudes, which in turn predicted lower reports of corporal punishment use. Among fathers, higher education predicted lower endorsement of corporal punishment, which in turn predicted lower reports of its use. Results suggest that education has an indirect relation to use of corporal punishment through parenting cognitions, and highlight distinctions in Filipino mothers\u27 and fathers\u27 parenting roles